FNDC5-knock out induces skeletal muscle and liver metabolic disorders in mice, especially in mice with high fat diet

FNDC5-knock out induces skeletal muscle and liver metabolic disorders in mice, especially in mice with high fat diet | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article FNDC5-knock out induces skeletal muscle and liver metabolic disorders in mice, especially in mice with high fat diet Chao Xin, Yi Liu, Zheng Zhang, Jianan Wang, Hui Liu, Chengzhu Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6254178/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction It has been proved that FNDC5 plays a significant role in energy metabolism, myocardial protection and insulin sensitivity. However, the specific role and underlying pathways of FNDC5 in energy metabolism, including glucose and lipid metabolism and amino acid metabolism are unclear so far. Objectives This study objectives were to investigate the metabolic consequences of FDNC5 knockout in mouse muscle and liver tissue. Methods Wildtype (WT) and KO mice were fed either normal or high fat diets (HFD), and then global biochemical profiles were determined in gastrocnemius muscle and liver, which aims to examine possible interactions between FDNC5 function and dietary composition. Results Knockout of the FDNC5 gene resulted in a substantial number of metabolite differences, relative to WT in both muscle and liver. Metabolic differences between KO and WT tissues were much more predominant in the high fat diet mice. The data suggest decreased utilization of glucose for glycolysis in HFD KO muscle, along with a possible increase in glycogen synthesis. Glycolysis might be reduced in HFD KO muscle. Most amino acids were elevated in HFD FNDC5 KO muscle. FNDC5 KO raised the levels of liver glucose 6-phosphate and phosphoenolpyruvate, relative to WT, under HFD condition. Glycogen degradation may be reduced in FNDC5 KO livers. FNDC5 KO reduced succinate and increases fumarate under HFD conditions. Liver amino acid levels were elevated in HFD FNDC5 KO livers relative to HFD WT. Conclusion Metabolomic profiling revealed numerous changes to muscle and liver metabolites that suggest broad-ranging effects of FNDC5 on tissue metabolism. The data suggests a number of metabolic pathways to explore further. Health sciences/Endocrinology/Endocrine system and metabolic diseases/Diabetes Health sciences/Endocrinology/Endocrine system and metabolic diseases/Obesity FNDC5 metabolic disorders metabolomic profile skeletal muscle liver Figures Figure 1 Figure 2 Figure 3 Introduction Cardiovascular diseases are the leading cause of death globally nowadays, threatening human health 1 . Many risk factors, such as dyslipidemia, smoking, obesity, and the lack of physical activity, contribute to the occurrence of cardiovascular diseases 2 . The occurrence and development of these diseases are closely related to the metabolism of sugar, fatty acid and amino acid. The maintenance of metabolic balance is a complex and delicate process involving multiple organs, tissues and cellular metabolic pathways and many signal molecules and regulatory factors. Skeletal muscle and liver serve as majors for metabolism including glucose, fatty acid and amino acids. As an important protein molecule, FNDC5/irisin has caused wide concern in the metabolic field in recent years. The protein FNDC5 (fibronectin type III domain–containing 5 protein contains a signal peptide, a hydrophobic transmembrane domain, a fibronectin III domain (the main part of irisin in the extracellular), and a carboxyterminal domain in the cytoplasm 3 . Irisin is a cleaved product from FNDC5 by peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α)-mediated proteolysis in a variety of human tissues such as liver, brain, adipose, muscle, and heart 4 , 5 . Aerobic exercise or cold-induced shivering could upregulate the expression of FNDC5/irisin, which induces the browning of white fat by increasing the expression of thermogenic genes such as p38, PGC-1α and UCP-1 4,6,7 . Studies showed that patients with type 2 diabetes mellitus, metabolic syndrome and obesity got lower serum irisin level 8 – 10 . FNDC5/irisin regulates glucose metabolism, promoting the uptake and utilization of glucose, plays a key role in lipid metabolism, regulating the synthesis and catabolic process of fat, and promotes amino acid uptake in muscle cells 11 , 12 . Although it is well established that FNDC5/irisin exerts beneficial metabolic effects, further investigations need to be conducted to better understand the underlying molecular mechanisms. Metabolomics is now emerging as a powerful tool for providing a precise functional profile of cellular biochemistry, and contributes to improved diagnostics, preventative strategies, and treatments for patients today 13 . In this study, we used FND5KO mice to explore the impacts of dietary high fat content and FNDC5 on global metabolic profile in skeletal muscle and liver, and correlate these with biochemical data and whole-body glucose homeostasis. Materials and methods All animal experiments were performed according to the National Institutes of Health Guidelines on the Use of Laboratory Animals, and were approved by the Fourth Military Medical University Committee on Animal Care. We confirm that the present study has been reported in full compliance with the ARRIVE guidelines. All aspects of the experimental design, implementation, and reporting, have been carried out in accordance with these guidelines. FNDC5 knockout mice Irisin is produced by proteolytic processing of a transmembrane receptor. Fndc5 is a 209-residue protein with an N-terminal 29-residue signal sequence followed by the irisin or putative fibronectin III domain, a linking peptide, a transmembrane domain, and a 39-residue cytoplasmic segment. Cleavage in the linking peptide region releases soluble irisin into the extracellular milieu. Therefore, we knocked out the Fndc5 gene to generate irisin lacking mice. Female Fndc5-heterozygous (+/−) mice were generated by Model Animal Research Center (Nanjing, China). Transcription activator-like effector nuclease (TALEN) technology was used to shear the DNA encoding the exon of the target gene. Fndc5 has six exons, and exon 2 of the coding region of Fndc5 gene was cut by TALEN nuclease, and the frameshift was realized by self-repair, resulting in premature termination of protein translation and termination of translation, thereby knocking out the gene. Fndc5-heterozygous (+/−) female mice were bred with C57BL/6 WT male mice (Experimental Animal Center, Fourth Military Medical University) to produce heterozygous (+/−) male mice. Subsequently, female Fndc5-deficient (−/−) mice (Fig. 1 C) were created by mating heterozygous (+/−) female mice with heterozygous (+/−) male mice. PCR-based genotyping analysis with tail genomic DNA was performed for Fndc5 using the following pr-imers: 5′-ACTAGCTCTCGCCTTGGTTCAAG − 3′ (forward) and 5′-TGGCTGACCTTGGACTTGAAGTC − 3′ (reverse). Animals and experimental protocols Male C57/BL6 mice at the age of 4–6 weeks were purchased from the Laboratory Animal Center of the Air Force Medical University, and maintained in a temperature-controlled barrier facility with a 12-hour light/dark cycle. Mice were fed with rodent chow until the age of 8–10 weeks. All animals were given access to food and water. WT or FNDC5-KO mice were fed either normal diet or the 60% high fat diet at the age of 6 weeks for a further 6weeks, resulting in four groups: WT-ND, WT-HFD, FNDC5-KO ND, and FNDC5-KO HFD. A few HFD-fed FNDC5-KO mice were given irisin (0.5 µg/g, irisin/body weight, Abnova, Taiwan, China)(a dose determined from previous report 7 )twice a day for an additional 2 weeks after the initial 6-week HFD feeding period and compared with untreated HFD-fed FNDC-5 KO mice, with chow-fed FNDC-5-KO mice as the genotype-diet control. Then skeletal muscle and liver tissues were isolated for further investigation. Western blot analysis Proteins were separated on SDS-PAGE gels, transferred to PVDF (polyvinylidenedifluoride, Millipore), and incubated with antibodies against, Akt (1:1000, Abcam), phospho-Akt (T308, 1:1000, Abcam), phospho-Akt (S473, 1:1000, Abcam),, and β-actin (1:1,000, Santa Cruz Biotechnology) overnight at 4 ℃. After washing blots to remove excessive primary antibody binding, the blots were incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000, abgent). Antibody binding was detected using enhanced chemiluminescence (Millipore). Film was scanned with ChemiDocXRS (Bio-Rad Laboratory, Hercules, CA). Immunoblot band intensity was analyzed using Lab Image software. Hyperinsulinemic-euglycemic clamp Hyperinsulinemic-euglycemic clamp was performed as described before 14 . Serum samples collected at experimental time = 0, 75, 80, 85, 90, 95, 100, 105, 110, 115, and 120 min were used to determine the Rd, Ra. Glucose infusion rate (GIR, mg/kg/min) during the last 35 min of the clamp (steady-state) was used to assess whole body insulin sensitivity. Whole-body insulin-mediated glucose disposal (Rd) was calculated by modified Steele’s single pool steady-state equations. The difference between Rd and GIR yields the rate of endogenous glucose production (EGP). Hepatic insulin sensitivity is given as percentage of insulin-mediated suppression of EGP vs basal conditions, whereas whole-body insulin sensitivity is represented as clamp Rd normalized to steady-state insulinemia 14 . Intraperitoneal glucose tolerance test (IPGTT) For the glucose tolerance test, the animals were starved 5–6 h before receiving a bolus injection of glucose (2 g/kg body wt i.p.). Tail-vein blood samples were collected after 15, 30, 60, and 120 min, and blood glucose level (millimoles per liter) was determined with a glucometer (Conture; Bayer). Circulating free fatty acid and triglyceride assay Circulating free fatty acid and triglyceride levels were assessed using free fatty acid assay kit and triglyceride assay kit (Abcam). Metabolomic analysis Samples (skeletal muscle and liver) were stored at -80℃. Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Then global biochemical profiles were determined in gastrocnemius muscle and liver as fee for service by Metabolon as previously described 15 . Statistics analyses Data in Fig. 1 were presented as mean ± SEM and the differences among three or more groups were analyzed by ANOVA. Western blot densities were analyzed with the Kruskal-Wallis test followed by a Dunn post hoc test. ANOVA contrasts were used to identify biochemicals that differed significantly between experimental groups. A P value < 0.05 was considered statistically significant. All statistical tests were performed using GraphPad Prism software version 5.0 (GraphPad Software, San Diego, CA). Results Result 1 Changes in whole-body glucose metabolism induced by HFD with or without irisin There was an exaggerated development of whole-body insulin resistance in FNDC5-KO mice in response to an HFD, indicated by glucose tolerance tests (Fig. 2 A). We then sought to determine whether restoration of irisin in FNDC5-KO mice would significantly revert HFD-induced metabolic deterioration. We also performed more detailed analyses of whole-body insulin sensitivity and glucose homeostasis using the hyperinsulinemiceuglycemic clamp to determine whether irisin correct HFD-induced insulin resistance. Figure 2 B shows that HFD caused a significant decrease in glucose infusion rate and that irisin treatment partially and significantly reversed this defect. Insulin-stimulated whole-body glucose disposal (Rd), normalized to steady-state insulinemia, was significantly lower in HFD mice confirming severe skeletal muscle insulin resistance (Fig. 2 C). Likewise, insulin-mediated suppression of EGP was lower in HFD group upon normalization to steady-state insulinemia (Fig. 2 D). Phosphorylation of AKT T308 and AKT S473 was decreased in HFD FNDC5KO mice confirming insulin resistance in skeletal muscle and liver at the molecular level, and irisin treatment can indeed correct HFD-induced both whole-body and hepatic insulin resistance (Fig. 2 E, F, G, I, J, K). Although irisin treatment significantly improved HFD-induced changes in whole-body glucose homeostasis, the changes were not complete reversal, as the negative impact of HFD is so severe as to be dominant. The enhances of circulating free fatty acid and triglyceride level induced by HFG were corrected by irisin partly (Fig. 2 H, L). Result 2 Increased glucose levels in HFD FNDC5 KO muscle Glucose levels were markedly higher in HFD KO muscle, relative to all other groups (Table 1). Though glucose was higher, downstream glycolysis metabolites glucose 6-phosphate, fructose 6-phosphate, 3-phosphoglycerate and lactate were lower in HFD KO muscle relative to the other groups. While glucose 6-phosphate was reduced in HFD KO muscle, glucose 1-phosphate was increased. Glucose 1-phosphate is an intermediate in glycogen synthesis and its increase may indicate that the KO, in combination with a high fat diet, leads to increased glycogen synthesis. Glycogen does serve as an energy reserve in muscle tissue. Sorbitol was also increased in HFD KO muscle, which would be consistent with an increased flow of glucose into the sorbitol shunt pathway. Overall, the data suggest decreased utilization of glucose for glycolysis in HFD KO muscle, along with a possible increase in glycogen synthesis (Fig. 3 A). Result 3 TCA cycle metabolites downstream of citrate were elevated in HFD FNDC5 KO muscle FNDC5 KO resulted in substantial TCA cycle metabolite changes, in muscle, under HFD conditions (Table 1). Increases in TCA intermediates can reflect changes to inflow or outflow, though it can be difficult to draw definitive conclusions as to carbon flow through the cycle. Interestingly, citrate was decreased in HFD KO muscle, but other metabolites downstream of citrate were elevated, relative to WT and KO muscle from mice fed a normal diet. Cis-aconitate, derived from citrate, was strongly increased in HFD KO muscle, suggesting a possible increase in aconitase activity. Another possibility for the lower citrate levels in HFD KO muscle might be a reduction in citrate synthase activity, converting acetyl-CoA and oxaloacetate to citrate. Increased 3-hydroxybutyrate (BHBA), which can be generated through ketogenesis when acetyl-CoA is in excess, might support a hypothesis of lower uptake of acetyl-CoA into the TCA cycle, which may be consistent with an overall reduced carbon flux through the cycle (Fig. 3 B). Since it appeared that glycolysis might be reduced in HFD KO muscle, one might speculate that carbon entry into the TCA cycle may be more strongly supported by fatty acid β-oxidation, or amino acid anaplerosis. Result 4 Most amino acids were elevated in HFD FNDC5 KO muscle All muscle tissue amino acids were elevated in HFD KO animals, with the exception of glutamine (Table 1). Since essential amino acids, such as methionine and tryptophan, were elevated, along with non-essential amino acids, not all the elevations can be related to increased synthesis. The two most highly elevated amino acids in HFD KO muscle, were aspartate and asparagine. Glutamine was the only amino acid that was lower in HFD KO muscle, relative to muscle from the other groups. Result 5 FNDC5 KO raised the levels of liver glucose 6-phosphate and phosphoenolpyruvate, relative to WT, under HFD conditions In contrast to muscle tissue, FNDC5 KO increased some glycolysis intermediates, relative to WT when mice were fed a HFD (Table 2). Glucose 6-phosphate and phosphoenolpyruvate (PEP) were elevated to a statistically significant level in HFD KO liver, relative to HFD WT. Other glycolysis intermediates trended higher in the HFD KO to WT comparison. Glycolysis intermediates were generally reduced in WT HFD livers, relative to WT normal diet livers. The KO had little effect on liver glycolysis intermediates, when mice were fed a normal diet. Result 6 Glycogen degradation may be reduced in FNDC5 KO livers. Under both HFD and ND feeding conditions, maltose and the maltose multimers maltotetraose and maltotriose were reduced in KO livers relative to WT (Table 2). Maltose and the multimers are generated in the breakdown of glycogen, eventually leading to the production of glucose 6-phosphate for glycolysis, or glucose for entry into the blood stream. Lower levels of the maltose metabolites in KO livers may be an indication of decreased glycogen degradation, or perhaps a lack of glycogen stores (Fig. 3 C). Result 7 FNDC5 KO reduced succinate and increases fumarate under HFD conditions Levels of TCA cycle metabolites citrate and α-ketoglutarate were reduced in HFD livers, relative to ND livers (only α-KG significant to p < 0.05), as shown in Table 2. In HFD WT livers, succinate was elevated, relative to ND WT and HFD KO, while downstream TCA cycle metabolites fumarate and malate were lowest in the HFD WT livers. KO of FNDC5 appeared to modulate the effects of HFD on succinate, fumarate and malate. If muscle amino acids, which were elevated in HFD KO tissue, enter the blood stream, they might support liver TCA cycle activity. Result 8 Liver amino acid levels were elevated in HFD FNDC5 KO livers relative to HFD WT Study of amino acid metabolism by metabolomic profiling revealed a trendof significantly increased levels of most amino acids in HFD WT livers relative to ND WT and HFD KO (Table 2). The exception to the lower amino acids in HFD WT livers was glutamine, which curiously was also the exception for muscle in comparing HFD KO to all other muscle groups. Discussion In the present study, we identified precise changes in metabolic parameters in skeletal muscle and liver of FNDC5KO mice fed an HFD using metabolomic profiling. Here we first validated the changes in metabolism and insulin sensitivity in FNDC5KO mice with HFD by performing hyperinsulinemic-euglycemic clamp studies to assess whole-body and hepatic insulin sensitivity and glucose metabolism. Several previous studies have shown that HFG could induce exaggerated insulin resistance in mice, and irisin could improve glucose uptake of muscle 8 – 11 , and this study showed that FNDC5KO mice got glucose/lipid metabolic derangements, reduced glucose tolerance, and insulin resistance. These results were consistent with previous studies showing that irisin overexpression improves glucose/lipid metabolism and insulin resistance in HFD mice 16 . The influence of irisin on metabolism correlated with improved activation of Akt in skeletal muscle and liver. Irisin also enhanced removal of FFA and TG from the circulation. Metabolic profiling has been widely used in several studies including diabetes-related studies 15 , 17 – 22 . For example, in a study of mice after 12 weeks on an HFD, HFD-induced disturbances and effects of adiponectin on the disturbances in muscle metabolomic profile were reported. Similarly, to gain insight into precisely where metabolic changes occurred in the FNDC5KO mice with ND or HFD, we used an untargeted metabolomic profiling platform to analyze intermediate metabolites involved in glucose, fatty acid, and amino acid metabolism 15 . According the available metabolomic data, we focused on the most statistically significant changes and offered a few observations form a cursory view of the data. Comparison of global biochemical profiles for muscle and liver tissue from KO and WT mice revealed several key metabolic differences. Many studies have shown that irisin plays an important role in glucose homeostasis both in physiological and pathological conditions. Several prospective studies evaluated the significant associations between lipid metabolites and risk of type 2 diabetes 23 – 25 . Skeletal muscle secretome contributes to the maintenance and regulation of overall physiological health through its paracrine and endocrine functions. The treatment of skeletal muscle cells with irisin increased uptake of glucose and fatty acid. The irisin treatment regulated glucose and lipid metabolism in myocytes by upregulating the expression of genes involved in glucose transport and lipid metabolism and suppressing the expression of genes that are involved in glycogenolysis or gluconeogenesis 26 . In hepatocytes, treatment with irisin reduces gluconeogenesis and stimulates glycogenesis through the phosphoinositide 3-kinase (PI3K)–AKT pathway 27 . And intraperitoneal injection of irisin in diabetic C57BL/6 mice decreased PEPCK and G6p expression in the liver by activating the AMPK pathway 11 . Consistent with previous studies, our data showed that downstream glycolysis metabolites glucose 6-phosphate, fructose 6-phosphate, 3-phosphoglycerate and lactate were lower, while glucose 1-phosphate was increased in HFD KO muscle relative to the other groups. This study indicates FNDC5KO with HFD decreased utilization of glucose for glycolysis, and increased in glycogen. In contrast to muscle tissue, FNDC5 KO increased some glycolysis intermediates, relative to WT when mice were fed a HFD synthesis in HFD KO muscle. Decreased glucose along with increased glucose 6-P and PEP in HFD KO mice relative to HFD WT mice, may suggest increased entry of glucose into the glycolysis pathway in liver. Glucose and glucose 6-P might also be consumed for glycogen synthesis, though glucose 1-P was not measured in liver. Maltose and the maltose multimers maltotetraose and maltotriose were reduced in KO livers relative to WT, which may indicate decreased glycogen degradation, or perhaps a lack of glycogen stores in liver. Irisin plays a role in energy balance in different tissues regulates metabolism. Dysregulation of FNDC5 may lead to the start of metabolic disorders. It has been shown that FNDC5/irisin could increase thermogenesis in WAT by increasing oxygen consumption and energy expenditure 5 , 7 . A study showed that FNDC5 expression was highly related to basic muscle fiber composition, and was necessary for a healthy metabolic state. moderately increased in irisin values strongly increase energy expenditure, reduce BW, and alleviate diet-induced insulin resistance 5 , 28 , 29 . The mitochondrial TCA, or Krebs, cycle is primarily noted to link the catabolism of carbohydrates, lipids and some amino acids to ATP production via supplying reducing equivalents through oxidative phosphorylation. Our data showed that TCA cycle metabolites downstream of citrate were elevated in HFD FNDC5 KO muscle. FNDC5 KO reduced succinate and increases fumarate in liver under HFD condition. Increases in succinate, with decreased levels of fumarate, may suggest some level of decrease in succinate dehydrogenase (SDH) activity. BHBA was marginally affected by diet and genotype, with an elevation in ND WT levels being driven by a potential outlier. Liver TCA cycle metabolites were modulated by diet and genotype, to some degree, but differences between groups were less pronounced than what was observed in muscle. Studies indicated that changes in BCAA profiles were linked with insulin resistance, obesity and diabetes 30 , 31 . Several studies showed that BCAAs supplementation could impair glucose metabolism and insulin sensitivity while restriction of BCAAs could improve metabolic health 32 – 35 . Few studies have investigated the link between irisin and amino acid metabolism, especially BCAA metabolism. Our metabolomic profiling highlighted an interesting change in amino acid metabolism. All muscle tissue amino acids were elevated in HFD KO animals, with the exception of glutamine. Increased amino acid levels may result from increased uptake from the blood stream, increased synthesis, increased protein degradation, or a decline in utilization for protein synthesis. The two most highly elevated amino acids in HFD KO muscle, were aspartate and asparagine. Aspartate can be synthesized from oxaloacetate and asparagine is synthesized from aspartate. Elevation of aspartate could be tied in with the decline in citrate levels. If more oxaloacetate goes toward aspartate synthesis, this might lead to a decline in citrate production and increased amounts of acetyl-CoA going into 3-hydroxybutyrate (BHBA) production. Alternatively, a reduction in citrate synthase activity could increase oxaloacetate levels (not measured) resulting in shunting into aspartate production. Glutamine was the only amino acid that was lower in HFD KO muscle, relative to muscle from the other groups. Some glutamine may be consumed in the conversion of aspartate to asparagine, but glutamine can also be utilized in many other metabolic reactions, including glutaminolysis (see Slide 9 pathway schematic). Amino acids can be used to support TCA cycle function through anaplerosis and an increase in muscle amino acids may reflect increased mobilization for this purpose, or a decrease in consumption for support of oxidative phosphorylation. Consistent with the results in muscle, liver amino acid levels were elevated in HFD FNDC5 KO livers relative to HFD WT. A reduction in glutaminolysis might limit decreases in glutamine levels, but glutamine is utilized in other metabolic pathways as well. Under HFD conditions, the KO could be interpreted as attenuating the reduction in liver amino acids occurring for HFD WT vs. ND WT. Differences in liver HFD WT vs. HFD KO amino acid levels could result from increased amino acid uptake from the bloodstream or decreased utilization for protein synthesis or anaplerotic support for oxidative phosphorylation. Knockout of FNDC5 resulted in a large number of metabolite changes in both muscle and liver, with the greatest effects observed for KO muscle tissue in mice fed the high fat diet. This report focused on energy-related metabolic pathways, given the reported role of FNDC5-derived irisin in modulating WAT metabolism to increase thermogenic oxidative phosphorylation. FNDC5 KO had strong effects on muscle metabolism, especially under HFD conditions. These KO-dependent muscle metabolite changes would seem to indicate that FNDC5’s impact on muscle tissue go beyond serving as a FNDC5 repository to generate irisin in response to exercise. The effect of the KO was especially strong in HFD mice, perhaps suggesting a role for FNDC5 in lipid metabolism. The FNDC5 KO appeared to alter glucose metabolism and TCA cycle function, in both muscle and liver. This was more clearly evident in the HFD animals. The effects of the KO on amino acid levels in both muscle and liver are intriguing. Muscle can supply amino acids to liver and other tissues, to meet energy needs under fasting conditions, but these mice were not fasted. Muscle can also supply amino acids during periods of prolonged exercise. It is not clear if the amino acid elevations in HFD KO muscle and reduced decline in HFD KO liver, relative to HFD WT, are related to FNDC5 modulation of amino acid mobilization, or other aspects of amino acid metabolism. Another area of interest not covered in this report are lipid metabolites. Many changes in lipid metabolites were more closely tied to diet, than to genotype, but there also appeared to be some genotype-associated changes. In summary, metabolomic profiling revealed numerous changes to muscle and liver metabolites that suggest broad ranging effects of FNDC5 on tissue metabolism. The data suggests a number of metabolic pathways to explore further. Declarations Data sharing statement All data presented in this manuscript are available from the corresponding author upon request. Conflict of Interest The authors declare that there is no duality of interest associated with this manuscript. Acknowledgements The work presented here was carried out in collaboration between all authors. Ling Tao and Zhitao Jin defined the research theme and revised the manuscript critically. Chao Xin, Yi Liu and Zheng Zhang designed methods and experiments, carried out the laboratory experiments, and wrote the paper. Jianan Wang, Hui Liu, Chengzhu Wang, Lijuan Zhang, Chengrong Zheng and Xiang Liao collected and analyzed the data, interpreted the results. References Pinto, F. J. et al. World Heart Day 2021: COVID-19, digital health, and tackling cardiovascular disease. Lancet 398 , 1467-1468, doi:10.1016/S0140-6736(21)02144-9 (2021). Yusuf, S. et al. 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Increased levels of plasma acylcarnitines in obesity and type 2 diabetes and identification of a marker of glucolipotoxicity. Obesity (Silver Spring) 18 , 1695-1700, doi:10.1038/oby.2009.510 (2010). Shah, H. et al. BCAAs acutely drive glucose dysregulation and insulin resistance: role of AgRP neurons. Nutr Diabetes 14 , 40, doi:10.1038/s41387-024-00298-y (2024). Jang, C. et al. A branched-chain amino acid metabolite drives vascular fatty acid transport and causes insulin resistance. Nat Med 22 , 421-426, doi:10.1038/nm.4057 (2016). Zhang, F. et al. Branched Chain Amino Acids Cause Liver Injury in Obese/Diabetic Mice by Promoting Adipocyte Lipolysis and Inhibiting Hepatic Autophagy. EBioMedicine 13 , 157-167, doi:10.1016/j.ebiom.2016.10.013 (2016). Solon-Biet, S. M. et al. Branched chain amino acids impact health and lifespan indirectly via amino acid balance and appetite control. Nat Metab 1 , 532-545, doi:10.1038/s42255-019-0059-2 (2019). Richardson, N. E. et al. Lifelong restriction of dietary branched-chain amino acids has sex-specific benefits for frailty and lifespan in mice. Nat Aging 1 , 73-86, doi:10.1038/s43587-020-00006-2 (2021). Tables Table 1. Metabolism analysis in skeletal muscle. Analyses were performed on skeletal muscle from WT or FNDC5 KO mice treated as described. Muscle Two-Way ANOVA Fold of Change ANOVA Contrasts Biochemical Name FNDC5 KO/WT HFD/ND Genotype Main Effect Diet Main Effect Genotype:Diet Interaction ND HFD WT FNDC5 KO sorbitol p≤0.05 p≤0.05 p≤0.05 1.01 83.63* 1.13 94.19* glucose p≥0.1 p≤0.05 p≤0.05 0.87 2.72* 1.16 3.65* Glucose 6-phosphate p≤0.05 p≤0.05 p≤0.05 2.23 0.04* 3.93 0.08* Glucose 1-phosphate p≤0.05 p≤0.05 0.05<p<0.10 1.35 4.86* 2.01 7.24* fructose-6-phosphate p≤0.05 p≤0.05 p≤0.05 2.10 0.13* 3.38 0.21* 3-phosphoglycerate p≤0.05 p≥0.1 0.05<p<0.10 0.67 0.31* 1.56 0.72* lactate p≤0.05 p≤0.05 p≤0.05 0.83 0.40* 1.01 0.49* citrate p≤0.05 p≥0.1 p≥0.1 0.68 0.48* 0.90 0.64 # cis-aconitate p≤0.05 p≤0.05 p≤0.05 0.82 4.38* 0.91 4.82* 3-hydroxybutyrate (BHBA) p≥0.1 p≥0.1 p≤0.05 0.45 3.04* 0.50 3.39* malate p≤0.05 p≤0.05 p≤0.05 1.23 3.26* 1.14 3.02* fumarate p≤0.05 p≤0.05 p≤0.05 1.08 2.42* 1.15 2.56* glycine p≤0.05 p≤0.05 p≤0.05 0.71 4.60* 0.63 4.09* serine p≤0.05 p≤0.05 p≤0.05 1.03 4.47* 1.08 4.69* threonine p≤0.05 p≤0.05 p≤0.05 1.17 2.72* 1.28 2.98* alanine p≤0.05 p≤0.05 p≤0.05 0.99 2.81* 1.22 3.44* aspartate p≤0.05 p≤0.05 p≤0.05 0.97 15.42* 0.62 9.90* asparagine p≤0.05 p≤0.05 p≤0.05 0.97 18.46* 1.08 20.53* glutamate p≤0.05 p≤0.05 p≤0.05 1.02 1.73* 1.14 1.93* glutamine 0.05<p<0.10 p≤0.05 p≤0.05 1.03 0.79* 1.01 0.78* histidine p≤0.05 p≤0.05 p≤0.05 1.06 4.29* 1.03 4.19* lysine p≤0.05 p≤0.05 p≤0.05 1.14 3.09* 1.37 3.72* phenylalanine p≤0.05 p≤0.05 p≤0.05 1.12 2.58* 1.17 2.70* tyrosine p≤0.05 p≤0.05 p≤0.05 1.04 2.34* 1.06 2.37* tryptophan p≤0.05 p≤0.05 p≤0.05 1.00 2.99* 0.97 2.91* leucine p≤0.05 p≤0.05 p≤0.05 0.93 4.01* 0.97 4.18* isoleucine p≤0.05 p≤0.05 p≤0.05 0.93 3.33* 0.96 3.46* valine p≤0.05 p≤0.05 p≤0.05 0.93 4.01* 0.97 4.18* methionine p≤0.05 p≤0.05 p≤0.05 0.95 3.43* 1.13 4.07* cysteine p≤0.05 p≤0.05 p≤0.05 0.90 4.08* 0.97 4.37* arginine p≤0.05 p≤0.05 p≤0.05 0.95 2.16* 0.99 2.26* proline p≤0.05 p≤0.05 p≤0.05 1.05 4.04* 1.27 4.89* * indicates significant difference (p≤0.05) between the groups shown # : narrowly missed statistical cutoff for significance 0.05<p<0.10 Table 2. Metabolism analysis in liver. Analyses were performed on liver from WT or FNDC5 KO mice treated as described. Liver Two-Way ANOVA Fold of Change ANOVA Contrasts Biochemical Name FNDC5 KO/WT HFD/ND Genotype Main Effect Diet Main Effect Genotype:Diet Interaction ND HFD WT FNDC5 KO glucose p≥0.1 p≤0.05 p≥0.1 0.99 0.90 0.89 0.81* glucose 6-phosphate p≤0.05 p≤0.05 p≤0.05 1.22 3.08* 0.29* 0.72 2-phosphoglycerate p≥0.1 p≤0.05 p≥0.1 0.90 1.87 0.39* 0.80 3-phosphoglycerate p≥0.1 p≤0.05 p≥0.1 0.91 1.35 0.55* 0.81 phosphoenolpyruvate (PEP) p≥0.1 p≤0.05 p≤0.05 0.80 2.36* 0.26* 0.76 maltotetraose p≤0.05 p≤0.05 p≥0.1 0.31* 0.28* 0.49* 0.44* maltotriose p≤0.05 p≤0.05 p≤0.05 0.28* 0.04* 0.50 0.07* maltose p≤0.05 p≤0.05 p≤0.05 0.54 0.06* 0.76 0.08* citrate p≥0.1 p≤0.05 p≥0.1 0.72 0.35 0.34 # 0.16 alpha-ketoglutarate p≤0.05 p≤0.05 0.05<p<0.10 0.20* 0.72 0.12* 0.43 succinate p≥0.1 p≤0.05 p≤0.05 1.08 0.59* 2.67* 1.46 # fumarate p≥0.1 0.05<p<0.10 p≤0.05 0.76 # 1.45* 0.60* 1.15 malate p≥0.1 p≥0.1 p≤0.05 0.84 1.39* 0.71* 1.18 3-hydroxybutyrate (BHBA) p≥0.1 p≥0.1 0.05<p<0.10 0.33 # 1.43 0.47 2.02 glycine p≤0.05 p≤0.05 p≤0.05 0.95 1.89* 0.50* 0.99 serine p≤0.05 p≤0.05 p≤0.05 1.02 2.19* 0.49* 1.05 threonine p≤0.05 p≤0.05 0.05<p<0.10 1.08 1.52* 0.70* 0.99 alanine 0.05<p<0.10 p≥0.01 p≤0.05 0.96 1.40* 0.74* 1.07 aspartate p≤0.05 p≤0.05 p≤0.05 1.03 2.63* 0.41* 1.06 asparagine 0.05<p<0.10 p≥0.1 p≤0.05 0.95 2.49* 0.51 # 1.34 glutamate p≥0.1 p≤0.05 p≤0.05 0.66 1.96* 0.36* 1.07 glutamine p≥0.1 p≤0.05 p≤0.05 1.43 0.75 2.24* 1.17 histidine p≤0.05 p≤0.05 p≤0.05 1.55* 3.49* 1.10 2.47* lysine p≤0.05 p≤0.05 p≤0.05 0.95 1.72* 0.60* 1.08 phenylalanine p≤0.05 p≤0.05 p≤0.05 1.14 2.09* 0.56* 1.02 tyrosine p≤0.05 p≥0.01 p≥0.1 1.48 # 1.97* *0.66 *0.87 tryptophan p≤0.05 p≤0.05 p≤0.05 1.20 1.95* 0.58* 0.95 leucine p≤0.05 p≤0.05 p≤0.05 1.04 2.27* 0.51* 1.11 isoleucine p≤0.05 p≤0.05 p≤0.05 1.10 2.28* 0.50* 1.03 valine p≤0.05 p≤0.05 p≤0.05 0.92 1.98* 0.52* 1.12 methionine p≤0.05 p≤0.05 p≤0.05 1.08 2.27* 0.48* 1.01 cysteine p≤0.05 p≥0.1 p≤0.05 0.93 1.82* 0.65* 1.26 arginine p≥0.1 0.05<p<0.10 0.05<p<0.10 0.58 1.41 0.30* 0.74 proline p≤0.05 0.05<p<0.10 p≤0.05 1.11 1.82* 0.63* 1.03 * indicates significant difference (p≤0.05) between the groups shown # : narrowly missed statistical cutoff for significance 0.05<p<0.10 Additional Declarations No competing interests reported. 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A, B: Schematic representation of the gene targeting procedure. The black arrowhead indicates the target exon as well as the blue lightning-like sign does. C: Confirmation of FNDC5\u003cem\u003e \u003c/em\u003eknockout by western blot.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6254178/v1/061e85cc6ba6c052e1e1083e.jpg"},{"id":79906996,"identity":"52e49298-371b-4825-bc88-fa3d149e7a3f","added_by":"auto","created_at":"2025-04-04 11:08:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":95610,"visible":true,"origin":"","legend":"\u003cp\u003eGlucose homeostasis, insulin sensitivity and related lipid analysis. A: IPGTT curves (millimoles per liter). B: Glucose infusion rate (GIR) (milligrams per kilogram per minute). C: Whole-body insulin sensitivity given as clamp Rd (mg/kg/min), normalized to steady-state insulinemia. D: Hepatic insulin sensitivity given as percentage of insulin mediated suppression of EGP vs basal conditions (normalized to steady-state insulinemia). E, F, G: Ratio of phosphorylated AKT (T308 and S473) to total AKT in skeletal muscle improved by Western Blot. H: Circulating FFA (millimoles per liter). H, I, J: Ratio of phosphorylated AKT (T308 and S473) to total AKT in liver improved by Western Blot. K: Circulating triacylglycerol (millimoles per liter). All values are presented as mean ± SEM. n=8/group. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01 contrasts with WT group.\u003csup\u003e # \u003c/sup\u003eP\u0026lt;0.05 contrasts with FNDC5 KO HFD group.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6254178/v1/83488a7724e898c4f556e2f2.jpg"},{"id":79907013,"identity":"7c9fc158-67f2-4cbd-86e0-ec9ff77d4064","added_by":"auto","created_at":"2025-04-04 11:09:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":68465,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolism process in organisms. A: Glucose metabolism. B: energy metabolism (TCA cycle). C: Glycogenolysis in liver.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6254178/v1/eb76168bd416f119334cbe5b.jpg"},{"id":82128460,"identity":"41496f54-e974-4663-82ec-7d3410bfeebf","added_by":"auto","created_at":"2025-05-07 04:46:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1459450,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6254178/v1/bb597dc1-2d7c-4aeb-8b74-f91ffa1941b0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"FNDC5-knock out induces skeletal muscle and liver metabolic disorders in mice, especially in mice with high fat diet","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular diseases are the leading cause of death globally nowadays, threatening human health\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Many risk factors, such as dyslipidemia, smoking, obesity, and the lack of physical activity, contribute to the occurrence of cardiovascular diseases \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The occurrence and development of these diseases are closely related to the metabolism of sugar, fatty acid and amino acid. The maintenance of metabolic balance is a complex and delicate process involving multiple organs, tissues and cellular metabolic pathways and many signal molecules and regulatory factors. Skeletal muscle and liver serve as majors for metabolism including glucose, fatty acid and amino acids. As an important protein molecule, FNDC5/irisin has caused wide concern in the metabolic field in recent years.\u003c/p\u003e \u003cp\u003eThe protein FNDC5 (fibronectin type III domain\u0026ndash;containing 5 protein contains a signal peptide, a hydrophobic transmembrane domain, a fibronectin III domain (the main part of irisin in the extracellular), and a carboxyterminal domain in the cytoplasm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Irisin is a cleaved product from FNDC5 by peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α)-mediated proteolysis in a variety of human tissues such as liver, brain, adipose, muscle, and heart\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Aerobic exercise or cold-induced shivering could upregulate the expression of FNDC5/irisin, which induces the browning of white fat by increasing the expression of thermogenic genes such as p38, PGC-1α and UCP-1\u003csup\u003e4,6,7\u003c/sup\u003e. Studies showed that patients with type 2 diabetes mellitus, metabolic syndrome and obesity got lower serum irisin level\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. FNDC5/irisin regulates glucose metabolism, promoting the uptake and utilization of glucose, plays a key role in lipid metabolism, regulating the synthesis and catabolic process of fat, and promotes amino acid uptake in muscle cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough it is well established that FNDC5/irisin exerts beneficial metabolic effects, further investigations need to be conducted to better understand the underlying molecular mechanisms. Metabolomics is now emerging as a powerful tool for providing a precise functional profile of cellular biochemistry, and contributes to improved diagnostics, preventative strategies, and treatments for patients today \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we used FND5KO mice to explore the impacts of dietary high fat content and FNDC5 on global metabolic profile in skeletal muscle and liver, and correlate these with biochemical data and whole-body glucose homeostasis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e All animal experiments were performed according to the National Institutes of Health Guidelines on the Use of Laboratory Animals, and were approved by the Fourth Military Medical University Committee on Animal Care. We confirm that the present study has been reported in full compliance with the ARRIVE guidelines. All aspects of the experimental design, implementation, and reporting, have been carried out in accordance with these guidelines.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFNDC5 knockout mice\u003c/h2\u003e \u003cp\u003eIrisin is produced by proteolytic processing of a transmembrane receptor. Fndc5 is a 209-residue protein with an N-terminal 29-residue signal sequence followed by the irisin or putative fibronectin III domain, a linking peptide, a transmembrane domain, and a 39-residue cytoplasmic segment. Cleavage in the linking peptide region releases soluble irisin into the extracellular milieu. Therefore, we knocked out the Fndc5 gene to generate irisin lacking mice. Female Fndc5-heterozygous (+/\u0026minus;) mice were generated by Model Animal Research Center (Nanjing, China). Transcription activator-like effector nuclease (TALEN) technology was used to shear the DNA encoding the exon of the target gene. Fndc5 has six exons, and exon 2 of the coding region of Fndc5 gene was cut by TALEN nuclease, and the frameshift was realized by self-repair, resulting in premature termination of protein translation and termination of translation, thereby knocking out the gene. Fndc5-heterozygous (+/\u0026minus;) female mice were bred with C57BL/6 WT male mice (Experimental Animal Center, Fourth Military Medical University) to produce heterozygous (+/\u0026minus;) male mice. Subsequently, female Fndc5-deficient (\u0026minus;/\u0026minus;) mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) were created by mating heterozygous (+/\u0026minus;) female mice with heterozygous (+/\u0026minus;) male mice. PCR-based genotyping analysis with tail genomic DNA was performed for Fndc5 using the following pr-imers: 5\u0026prime;-ACTAGCTCTCGCCTTGGTTCAAG \u0026minus;\u0026thinsp;3\u0026prime; (forward) and 5\u0026prime;-TGGCTGACCTTGGACTTGAAGTC \u0026minus;\u0026thinsp;3\u0026prime; (reverse).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals and experimental protocols\u003c/h3\u003e\n\u003cp\u003eMale C57/BL6 mice at the age of 4\u0026ndash;6 weeks were purchased from the Laboratory Animal Center of the Air Force Medical University, and maintained in a temperature-controlled barrier facility with a 12-hour light/dark cycle. Mice were fed with rodent chow until the age of 8\u0026ndash;10 weeks. All animals were given access to food and water. WT or FNDC5-KO mice were fed either normal diet or the 60% high fat diet at the age of 6 weeks for a further 6weeks, resulting in four groups: WT-ND, WT-HFD, FNDC5-KO ND, and FNDC5-KO HFD. A few HFD-fed FNDC5-KO mice were given irisin (0.5 \u0026micro;g/g, irisin/body weight, Abnova, Taiwan, China)(a dose determined from previous report\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e)twice a day for an additional 2 weeks after the initial 6-week HFD feeding period and compared with untreated HFD-fed FNDC-5 KO mice, with chow-fed FNDC-5-KO mice as the genotype-diet control. Then skeletal muscle and liver tissues were isolated for further investigation.\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eProteins were separated on SDS-PAGE gels, transferred to PVDF (polyvinylidenedifluoride, Millipore), and incubated with antibodies against, Akt (1:1000, Abcam), phospho-Akt (T308, 1:1000, Abcam), phospho-Akt (S473, 1:1000, Abcam),, and β-actin (1:1,000, Santa Cruz Biotechnology) overnight at 4 ℃. After washing blots to remove excessive primary antibody binding, the blots were incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000, abgent). Antibody binding was detected using enhanced chemiluminescence (Millipore). Film was scanned with ChemiDocXRS (Bio-Rad Laboratory, Hercules, CA). Immunoblot band intensity was analyzed using Lab Image software.\u003c/p\u003e\n\u003ch3\u003eHyperinsulinemic-euglycemic clamp\u003c/h3\u003e\n\u003cp\u003eHyperinsulinemic-euglycemic clamp was performed as described before\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Serum samples collected at experimental time\u0026thinsp;=\u0026thinsp;0, 75, 80, 85, 90, 95, 100, 105, 110, 115, and 120 min were used to determine the Rd, Ra. Glucose infusion rate (GIR, mg/kg/min) during the last 35 min of the clamp (steady-state) was used to assess whole body insulin sensitivity. Whole-body insulin-mediated glucose disposal (Rd) was calculated by modified Steele\u0026rsquo;s single pool steady-state equations. The difference between Rd and GIR yields the rate of endogenous glucose production (EGP). Hepatic insulin sensitivity is given as percentage of insulin-mediated suppression of EGP vs basal conditions, whereas whole-body insulin sensitivity is represented as clamp Rd normalized to steady-state insulinemia\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eIntraperitoneal glucose tolerance test (IPGTT)\u003c/h3\u003e\n\u003cp\u003eFor the glucose tolerance test, the animals were starved 5\u0026ndash;6 h before receiving a bolus injection of glucose (2 g/kg body wt i.p.). Tail-vein blood samples were collected after 15, 30, 60, and 120 min, and blood glucose level (millimoles per liter) was determined with a glucometer (Conture; Bayer).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCirculating free fatty acid and triglyceride assay\u003c/h2\u003e \u003cp\u003eCirculating free fatty acid and triglyceride levels were assessed using free fatty acid assay kit and triglyceride assay kit (Abcam).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMetabolomic analysis\u003c/h3\u003e\n\u003cp\u003eSamples (skeletal muscle and liver) were stored at -80℃. Samples were prepared using the automated MicroLab STAR\u0026reg; system from Hamilton Company. Then global biochemical profiles were determined in gastrocnemius muscle and liver as fee for service by Metabolon as previously described\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eStatistics analyses\u003c/h3\u003e\n\u003cp\u003eData in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and the differences among three or more groups were analyzed by ANOVA. Western blot densities were analyzed with the Kruskal-Wallis test followed by a Dunn post hoc test. ANOVA contrasts were used to identify biochemicals that differed significantly between experimental groups. A P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All statistical tests were performed using GraphPad Prism software version 5.0 (GraphPad Software, San Diego, CA).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eResult 1 Changes in whole-body glucose metabolism induced by HFD with or without irisin\u003c/h2\u003e \u003cp\u003eThere was an exaggerated development of whole-body insulin resistance in FNDC5-KO mice in response to an HFD, indicated by glucose tolerance tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We then sought to determine whether restoration of irisin in FNDC5-KO mice would significantly revert HFD-induced metabolic deterioration. We also performed more detailed analyses of whole-body insulin sensitivity and glucose homeostasis using the hyperinsulinemiceuglycemic clamp to determine whether irisin correct HFD-induced insulin resistance. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB shows that HFD caused a significant decrease in glucose infusion rate and that irisin treatment partially and significantly reversed this defect. Insulin-stimulated whole-body glucose disposal (Rd), normalized to steady-state insulinemia, was significantly lower in HFD mice confirming severe skeletal muscle insulin resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Likewise, insulin-mediated suppression of EGP was lower in HFD group upon normalization to steady-state insulinemia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Phosphorylation of AKT\u003csup\u003eT308\u003c/sup\u003e and AKT\u003csup\u003eS473\u003c/sup\u003e was decreased in HFD FNDC5KO mice confirming insulin resistance in skeletal muscle and liver at the molecular level, and irisin treatment can indeed correct HFD-induced both whole-body and hepatic insulin resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F, G, I, J, K). Although irisin treatment significantly improved HFD-induced changes in whole-body glucose homeostasis, the changes were not complete reversal, as the negative impact of HFD is so severe as to be dominant. The enhances of circulating free fatty acid and triglyceride level induced by HFG were corrected by irisin partly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, L).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eResult 2 Increased glucose levels in HFD FNDC5 KO muscle\u003c/h2\u003e \u003cp\u003eGlucose levels were markedly higher in HFD KO muscle, relative to all other groups (Table\u0026nbsp;1). Though glucose was higher, downstream glycolysis metabolites glucose 6-phosphate, fructose 6-phosphate, 3-phosphoglycerate and lactate were lower in HFD KO muscle relative to the other groups. While glucose 6-phosphate was reduced in HFD KO muscle, glucose 1-phosphate was increased. Glucose 1-phosphate is an intermediate in glycogen synthesis and its increase may indicate that the KO, in combination with a high fat diet, leads to increased glycogen synthesis. Glycogen does serve as an energy reserve in muscle tissue. Sorbitol was also increased in HFD KO muscle, which would be consistent with an increased flow of glucose into the sorbitol shunt pathway. Overall, the data suggest decreased utilization of glucose for glycolysis in HFD KO muscle, along with a possible increase in glycogen synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eResult 3 TCA cycle metabolites downstream of citrate were elevated in HFD FNDC5 KO muscle\u003c/h2\u003e \u003cp\u003eFNDC5 KO resulted in substantial TCA cycle metabolite changes, in muscle, under HFD conditions (Table\u0026nbsp;1). Increases in TCA intermediates can reflect changes to inflow or outflow, though it can be difficult to draw definitive conclusions as to carbon flow through the cycle. Interestingly, citrate was decreased in HFD KO muscle, but other metabolites downstream of citrate were elevated, relative to WT and KO muscle from mice fed a normal diet. Cis-aconitate, derived from citrate, was strongly increased in HFD KO muscle, suggesting a possible increase in aconitase activity. Another possibility for the lower citrate levels in HFD KO muscle might be a reduction in citrate synthase activity, converting acetyl-CoA and oxaloacetate to citrate. Increased 3-hydroxybutyrate (BHBA), which can be generated through ketogenesis when acetyl-CoA is in excess, might support a hypothesis of lower uptake of acetyl-CoA into the TCA cycle, which may be consistent with an overall reduced carbon flux through the cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Since it appeared that glycolysis might be reduced in HFD KO muscle, one might speculate that carbon entry into the TCA cycle may be more strongly supported by fatty acid β-oxidation, or amino acid anaplerosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eResult 4 Most amino acids were elevated in HFD FNDC5 KO muscle\u003c/h2\u003e \u003cp\u003eAll muscle tissue amino acids were elevated in HFD KO animals, with the exception of glutamine (Table\u0026nbsp;1). Since essential amino acids, such as methionine and tryptophan, were elevated, along with non-essential amino acids, not all the elevations can be related to increased synthesis. The two most highly elevated amino acids in HFD KO muscle, were aspartate and asparagine. Glutamine was the only amino acid that was lower in HFD KO muscle, relative to muscle from the other groups.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResult 5 FNDC5 KO raised the levels of liver glucose 6-phosphate and phosphoenolpyruvate, relative to WT, under HFD conditions\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn contrast to muscle tissue, FNDC5 KO increased some glycolysis intermediates, relative to WT when mice were fed a HFD (Table\u0026nbsp;2). Glucose 6-phosphate and phosphoenolpyruvate (PEP) were elevated to a statistically significant level in HFD KO liver, relative to HFD WT. Other glycolysis intermediates trended higher in the HFD KO to WT comparison. Glycolysis intermediates were generally reduced in WT HFD livers, relative to WT normal diet livers. The KO had little effect on liver glycolysis intermediates, when mice were fed a normal diet.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResult 6 Glycogen degradation may be reduced in FNDC5 KO livers.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUnder both HFD and ND feeding conditions, maltose and the maltose multimers maltotetraose and maltotriose were reduced in KO livers relative to WT (Table\u0026nbsp;2). Maltose and the multimers are generated in the breakdown of glycogen, eventually leading to the production of glucose 6-phosphate for glycolysis, or glucose for entry into the blood stream. Lower levels of the maltose metabolites in KO livers may be an indication of decreased glycogen degradation, or perhaps a lack of glycogen stores (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eResult 7 FNDC5 KO reduced succinate and increases fumarate under HFD conditions\u003c/h2\u003e \u003cp\u003eLevels of TCA cycle metabolites citrate and α-ketoglutarate were reduced in HFD livers, relative to ND livers (only α-KG significant to p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as shown in Table\u0026nbsp;2. In HFD WT livers, succinate was elevated, relative to ND WT and HFD KO, while downstream TCA cycle metabolites fumarate and malate were lowest in the HFD WT livers. KO of FNDC5 appeared to modulate the effects of HFD on succinate, fumarate and malate. If muscle amino acids, which were elevated in HFD KO tissue, enter the blood stream, they might support liver TCA cycle activity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResult 8 Liver amino acid levels were elevated in HFD FNDC5 KO livers relative to HFD WT\u003c/b\u003e \u003c/p\u003e \u003cp\u003eStudy of amino acid metabolism by metabolomic profiling revealed a trendof significantly increased levels of most amino acids in HFD WT livers relative to ND WT and HFD KO (Table\u0026nbsp;2). The exception to the lower amino acids in HFD WT livers was glutamine, which curiously was also the exception for muscle in comparing HFD KO to all other muscle groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we identified precise changes in metabolic parameters in skeletal muscle and liver of FNDC5KO mice fed an HFD using metabolomic profiling. Here we first validated the changes in metabolism and insulin sensitivity in FNDC5KO mice with HFD by performing hyperinsulinemic-euglycemic clamp studies to assess whole-body and hepatic insulin sensitivity and glucose metabolism. Several previous studies have shown that HFG could induce exaggerated insulin resistance in mice, and irisin could improve glucose uptake of muscle\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and this study showed that FNDC5KO mice got glucose/lipid metabolic derangements, reduced glucose tolerance, and insulin resistance. These results were consistent with previous studies showing that irisin overexpression improves glucose/lipid metabolism and insulin resistance in HFD mice\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The influence of irisin on metabolism correlated with improved activation of Akt in skeletal muscle and liver. Irisin also enhanced removal of FFA and TG from the circulation.\u003c/p\u003e \u003cp\u003eMetabolic profiling has been widely used in several studies including diabetes-related studies\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. For example, in a study of mice after 12 weeks on an HFD, HFD-induced disturbances and effects of adiponectin on the disturbances in muscle metabolomic profile were reported. Similarly, to gain insight into precisely where metabolic changes occurred in the FNDC5KO mice with ND or HFD, we used an untargeted metabolomic profiling platform to analyze intermediate metabolites involved in glucose, fatty acid, and amino acid metabolism\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. According the available metabolomic data, we focused on the most statistically significant changes and offered a few observations form a cursory view of the data. Comparison of global biochemical profiles for muscle and liver tissue from KO and WT mice revealed several key metabolic differences.\u003c/p\u003e \u003cp\u003eMany studies have shown that irisin plays an important role in glucose homeostasis both in physiological and pathological conditions. Several prospective studies evaluated the significant associations between lipid metabolites and risk of type 2 diabetes\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Skeletal muscle secretome contributes to the maintenance and regulation of overall physiological health through its paracrine and endocrine functions. The treatment of skeletal muscle cells with irisin increased uptake of glucose and fatty acid. The irisin treatment regulated glucose and lipid metabolism in myocytes by upregulating the expression of genes involved in glucose transport and lipid metabolism and suppressing the expression of genes that are involved in glycogenolysis or gluconeogenesis\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In hepatocytes, treatment with irisin reduces gluconeogenesis and stimulates glycogenesis through the phosphoinositide 3-kinase (PI3K)\u0026ndash;AKT pathway\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. And intraperitoneal injection of irisin in diabetic C57BL/6 mice decreased PEPCK and G6p expression in the liver by activating the AMPK pathway\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Consistent with previous studies, our data showed that downstream glycolysis metabolites glucose 6-phosphate, fructose 6-phosphate, 3-phosphoglycerate and lactate were lower, while glucose 1-phosphate was increased in HFD KO muscle relative to the other groups. This study indicates FNDC5KO with HFD decreased utilization of glucose for glycolysis, and increased in glycogen. In contrast to muscle tissue, FNDC5 KO increased some glycolysis intermediates, relative to WT when mice were fed a HFD synthesis in HFD KO muscle. Decreased glucose along with increased glucose 6-P and PEP in HFD KO mice relative to HFD WT mice, may suggest increased entry of glucose into the glycolysis pathway in liver. Glucose and glucose 6-P might also be consumed for glycogen synthesis, though glucose 1-P was not measured in liver. Maltose and the maltose multimers maltotetraose and maltotriose were reduced in KO livers relative to WT, which may indicate decreased glycogen degradation, or perhaps a lack of glycogen stores in liver.\u003c/p\u003e \u003cp\u003eIrisin plays a role in energy balance in different tissues regulates metabolism. Dysregulation of FNDC5 may lead to the start of metabolic disorders. It has been shown that FNDC5/irisin could increase thermogenesis in WAT by increasing oxygen consumption and energy expenditure\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. A study showed that FNDC5 expression was highly related to basic muscle fiber composition, and was necessary for a healthy metabolic state. moderately increased in irisin values strongly increase energy expenditure, reduce BW, and alleviate diet-induced insulin resistance\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The mitochondrial TCA, or Krebs, cycle is primarily noted to link the catabolism of carbohydrates, lipids and some amino acids to ATP production via supplying reducing equivalents through oxidative phosphorylation. Our data showed that TCA cycle metabolites downstream of citrate were elevated in HFD FNDC5 KO muscle. FNDC5 KO reduced succinate and increases fumarate in liver under HFD condition. Increases in succinate, with decreased levels of fumarate, may suggest some level of decrease in succinate dehydrogenase (SDH) activity. BHBA was marginally affected by diet and genotype, with an elevation in ND WT levels being driven by a potential outlier. Liver TCA cycle metabolites were modulated by diet and genotype, to some degree, but differences between groups were less pronounced than what was observed in muscle.\u003c/p\u003e \u003cp\u003eStudies indicated that changes in BCAA profiles were linked with insulin resistance, obesity and diabetes\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Several studies showed that BCAAs supplementation could impair glucose metabolism and insulin sensitivity while restriction of BCAAs could improve metabolic health\u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Few studies have investigated the link between irisin and amino acid metabolism, especially BCAA metabolism. Our metabolomic profiling highlighted an interesting change in amino acid metabolism. All muscle tissue amino acids were elevated in HFD KO animals, with the exception of glutamine. Increased amino acid levels may result from increased uptake from the blood stream, increased synthesis, increased protein degradation, or a decline in utilization for protein synthesis. The two most highly elevated amino acids in HFD KO muscle, were aspartate and asparagine. Aspartate can be synthesized from oxaloacetate and asparagine is synthesized from aspartate. Elevation of aspartate could be tied in with the decline in citrate levels. If more oxaloacetate goes toward aspartate synthesis, this might lead to a decline in citrate production and increased amounts of acetyl-CoA going into 3-hydroxybutyrate (BHBA) production. Alternatively, a reduction in citrate synthase activity could increase oxaloacetate levels (not measured) resulting in shunting into aspartate production. Glutamine was the only amino acid that was lower in HFD KO muscle, relative to muscle from the other groups. Some glutamine may be consumed in the conversion of aspartate to asparagine, but glutamine can also be utilized in many other metabolic reactions, including glutaminolysis (see Slide 9 pathway schematic). Amino acids can be used to support TCA cycle function through anaplerosis and an increase in muscle amino acids may reflect increased mobilization for this purpose, or a decrease in consumption for support of oxidative phosphorylation. Consistent with the results in muscle, liver amino acid levels were elevated in HFD FNDC5 KO livers relative to HFD WT. A reduction in glutaminolysis might limit decreases in glutamine levels, but glutamine is utilized in other metabolic pathways as well. Under HFD conditions, the KO could be interpreted as attenuating the reduction in liver amino acids occurring for HFD WT vs. ND WT. Differences in liver HFD WT vs. HFD KO amino acid levels could result from increased amino acid uptake from the bloodstream or decreased utilization for protein synthesis or anaplerotic support for oxidative phosphorylation.\u003c/p\u003e \u003cp\u003eKnockout of FNDC5 resulted in a large number of metabolite changes in both muscle and liver, with the greatest effects observed for KO muscle tissue in mice fed the high fat diet. This report focused on energy-related metabolic pathways, given the reported role of FNDC5-derived irisin in modulating WAT metabolism to increase thermogenic oxidative phosphorylation. FNDC5 KO had strong effects on muscle metabolism, especially under HFD conditions. These KO-dependent muscle metabolite changes would seem to indicate that FNDC5\u0026rsquo;s impact on muscle tissue go beyond serving as a FNDC5 repository to generate irisin in response to exercise. The effect of the KO was especially strong in HFD mice, perhaps suggesting a role for FNDC5 in lipid metabolism. The FNDC5 KO appeared to alter glucose metabolism and TCA cycle function, in both muscle and liver. This was more clearly evident in the HFD animals. The effects of the KO on amino acid levels in both muscle and liver are intriguing. Muscle can supply amino acids to liver and other tissues, to meet energy needs under fasting conditions, but these mice were not fasted. Muscle can also supply amino acids during periods of prolonged exercise. It is not clear if the amino acid elevations in HFD KO muscle and reduced decline in HFD KO liver, relative to HFD WT, are related to FNDC5 modulation of amino acid mobilization, or other aspects of amino acid metabolism. Another area of interest not covered in this report are lipid metabolites. Many changes in lipid metabolites were more closely tied to diet, than to genotype, but there also appeared to be some genotype-associated changes. In summary, metabolomic profiling revealed numerous changes to muscle and liver metabolites that suggest broad ranging effects of FNDC5 on tissue metabolism. The data suggests a number of metabolic pathways to explore further.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData sharing statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data presented in this manuscript are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no duality of interest associated with this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work presented here was carried out in collaboration between all authors. Ling Tao and Zhitao Jin defined the research theme and revised the manuscript critically. Chao Xin, Yi Liu and Zheng Zhang designed methods and experiments, carried out the laboratory experiments, and wrote the paper. Jianan Wang, Hui Liu, Chengzhu Wang, Lijuan Zhang, Chengrong Zheng and Xiang Liao collected and analyzed the data, interpreted the results.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePinto, F. J.\u003cem\u003e et al.\u003c/em\u003e World Heart Day 2021: COVID-19, digital health, and tackling cardiovascular disease. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e398\u003c/strong\u003e, 1467-1468, doi:10.1016/S0140-6736(21)02144-9 (2021).\u003c/li\u003e\n\u003cli\u003eYusuf, S.\u003cem\u003e et al.\u003c/em\u003e Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e364\u003c/strong\u003e, 937-952, doi:10.1016/S0140-6736(04)17018-9 (2004).\u003c/li\u003e\n\u003cli\u003eNie, Y., Dai, B., Guo, X. \u0026amp; Liu, D. 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J.\u003cem\u003e et al.\u003c/em\u003e Increased levels of plasma acylcarnitines in obesity and type 2 diabetes and identification of a marker of glucolipotoxicity. \u003cem\u003eObesity (Silver Spring)\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 1695-1700, doi:10.1038/oby.2009.510 (2010).\u003c/li\u003e\n\u003cli\u003eShah, H.\u003cem\u003e et al.\u003c/em\u003e BCAAs acutely drive glucose dysregulation and insulin resistance: role of AgRP neurons. \u003cem\u003eNutr Diabetes\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 40, doi:10.1038/s41387-024-00298-y (2024).\u003c/li\u003e\n\u003cli\u003eJang, C.\u003cem\u003e et al.\u003c/em\u003e A branched-chain amino acid metabolite drives vascular fatty acid transport and causes insulin resistance. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 421-426, doi:10.1038/nm.4057 (2016).\u003c/li\u003e\n\u003cli\u003eZhang, F.\u003cem\u003e et al.\u003c/em\u003e Branched Chain Amino Acids Cause Liver Injury in Obese/Diabetic Mice by Promoting Adipocyte Lipolysis and Inhibiting Hepatic Autophagy. \u003cem\u003eEBioMedicine\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 157-167, doi:10.1016/j.ebiom.2016.10.013 (2016).\u003c/li\u003e\n\u003cli\u003eSolon-Biet, S. M.\u003cem\u003e et al.\u003c/em\u003e Branched chain amino acids impact health and lifespan indirectly via amino acid balance and appetite control. \u003cem\u003eNat Metab\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 532-545, doi:10.1038/s42255-019-0059-2 (2019).\u003c/li\u003e\n\u003cli\u003eRichardson, N. E.\u003cem\u003e et al.\u003c/em\u003e Lifelong restriction of dietary branched-chain amino acids has sex-specific benefits for frailty and lifespan in mice. \u003cem\u003eNat Aging\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 73-86, doi:10.1038/s43587-020-00006-2 (2021).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eMetabolism analysis in skeletal muscle. Analyses were performed on skeletal muscle from WT or FNDC5 KO mice treated as described.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"727\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eMuscle\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" rowspan=\"3\" style=\"width: 231px;\"\u003e\n \u003cp\u003eTwo-Way ANOVA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 350px;\"\u003e\n \u003cp\u003eFold of Change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"35\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width: 350px;\"\u003e\n \u003cp\u003eANOVA Contrasts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"37\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eBiochemical\u003c/p\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 170px;\"\u003e\n \u003cp\u003eFNDC5 KO/WT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 180px;\"\u003e\n \u003cp\u003eHFD/ND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"37\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 80px;\"\u003e\n \u003cp\u003eGenotype Main Effect\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 66px;\"\u003e\n \u003cp\u003eDiet Main Effect\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 85px;\"\u003e\n \u003cp\u003eGenotype:Diet Interaction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"21\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eHFD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eWT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eFNDC5 KO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"32\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003esorbitol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e83.63*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e94.19*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003eglucose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.72*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e3.65*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003eGlucose 6-phosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.04*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e3.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.08*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003eGlucose 1-phosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.05\u0026lt;p\u0026lt;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e4.86*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e7.24*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003efructose-6-phosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.13*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e3.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.21*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e3-phosphoglycerate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.05\u0026lt;p\u0026lt;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.31*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.72*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003elactate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.40*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.49*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003ecitrate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.48*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.64\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003ecis-aconitate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e4.38*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e4.82*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e3-hydroxybutyrate (BHBA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e3.04*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e3.39*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003emalate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e3.26*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e3.02*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003efumarate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.42*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.56*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003eglycine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e4.60*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e4.09*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003eserine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e4.47*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e4.69*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003ethreonine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.72*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.98*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003ealanine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.81*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e3.44*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003easpartate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e15.42*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e9.90*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003easparagine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e18.46*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e20.53*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003eglutamate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.73*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e1.93*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003eglutamine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e0.05\u0026lt;p\u0026lt;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.79*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.78*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003ehistidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e4.29*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e4.19*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003elysine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e3.09*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e3.72*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003ephenylalanine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.58*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.70*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003etyrosine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.34*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.37*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003etryptophan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.99*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.91*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003eleucine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e4.01*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e4.18*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003eisoleucine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e3.33*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e3.46*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003evaline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e4.01*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e4.18*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003emethionine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e3.43*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e4.07*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003ecysteine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e4.08*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e4.37*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003earginine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.16*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.26*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003eproline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e4.04*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e4.89*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" valign=\"top\" style=\"width: 727px;\"\u003e\n \u003cp\u003e* indicates significant difference (p\u0026le;0.05) between the groups shown\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e#\u003c/sup\u003e: narrowly missed statistical cutoff for significance 0.05\u0026lt;p\u0026lt;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Metabolism analysis in liver. Analyses were performed on liver from WT or FNDC5 KO mice treated as described.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"728\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eLiver\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" rowspan=\"3\" style=\"width: 239px;\"\u003e\n \u003cp\u003eTwo-Way ANOVA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 350px;\"\u003e\n \u003cp\u003eFold of Change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"35\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width: 350px;\"\u003e\n \u003cp\u003eANOVA Contrasts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"37\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eBiochemical\u003c/p\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 170px;\"\u003e\n \u003cp\u003eFNDC5 KO/WT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 180px;\"\u003e\n \u003cp\u003eHFD/ND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"37\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 80px;\"\u003e\n \u003cp\u003eGenotype Main Effect\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 74px;\"\u003e\n \u003cp\u003eDiet Main Effect\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 85px;\"\u003e\n \u003cp\u003eGenotype:Diet Interaction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"21\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eHFD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eWT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eFNDC5 KO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"32\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003eglucose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.81*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003eglucose 6-phosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e3.08*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.29*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003e2-phosphoglycerate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.39*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003e3-phosphoglycerate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.55*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003ephosphoenolpyruvate (PEP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.36*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.26*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003emaltotetraose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.31*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.28*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.49*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.44*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003emaltotriose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.28*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.04*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.07*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003emaltose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.06*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.08*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003ecitrate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.34\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003ealpha-ketoglutarate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.05\u0026lt;p\u0026lt;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.20*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.12*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003esuccinate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.59*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.67*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.46\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003efumarate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e0.05\u0026lt;p\u0026lt;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.76\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.45*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.60*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003emalate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.39*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.71*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003e3-hydroxybutyrate (BHBA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.05\u0026lt;p\u0026lt;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.33\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003eglycine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.89*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.50*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003eserine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.19*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.49*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003ethreonine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.05\u0026lt;p\u0026lt;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.52*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.70*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003ealanine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e0.05\u0026lt;p\u0026lt;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026ge;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.40*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.74*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003easpartate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.63*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.41*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003easparagine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e0.05\u0026lt;p\u0026lt;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.49*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.51\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eglutamate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.96*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.36*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eglutamine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e2.24*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003ehistidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.55*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e3.49*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.47*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003elysine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.72*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.60*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003ephenylalanine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.09*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.56*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003etyrosine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026ge;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.48\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.97*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e*0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e*0.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003etryptophan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.95*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.58*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003eleucine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.27*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.51*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003eisoleucine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.28*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.50*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003evaline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.98*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.52*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003emethionine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2.27*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.48*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003ecysteine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.82*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.65*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003earginine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026ge;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e0.05\u0026lt;p\u0026lt;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.05\u0026lt;p\u0026lt;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.30*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003eproline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e0.05\u0026lt;p\u0026lt;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003ep\u0026le;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.82*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e0.63*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" valign=\"top\" style=\"width: 728px;\"\u003e\n \u003cp\u003e*\u0026nbsp;indicates significant difference (p\u0026le;0.05) between the groups shown\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e#\u003c/sup\u003e:\u0026nbsp;narrowly missed statistical cutoff for significance 0.05\u0026lt;p\u0026lt;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"FNDC5, metabolic disorders, metabolomic profile, skeletal muscle, liver","lastPublishedDoi":"10.21203/rs.3.rs-6254178/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6254178/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction\u003c/h2\u003e \u003cp\u003eIt has been proved that FNDC5 plays a significant role in energy metabolism, myocardial protection and insulin sensitivity. However, the specific role and underlying pathways of FNDC5 in energy metabolism, including glucose and lipid metabolism and amino acid metabolism are unclear so far.\u003c/p\u003e\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eThis study objectives were to investigate the metabolic consequences of FDNC5 knockout in mouse muscle and liver tissue.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWildtype (WT) and KO mice were fed either normal or high fat diets (HFD), and then global biochemical profiles were determined in gastrocnemius muscle and liver, which aims to examine possible interactions between FDNC5 function and dietary composition.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eKnockout of the FDNC5 gene resulted in a substantial number of metabolite differences, relative to WT in both muscle and liver. Metabolic differences between KO and WT tissues were much more predominant in the high fat diet mice. The data suggest decreased utilization of glucose for glycolysis in HFD KO muscle, along with a possible increase in glycogen synthesis. Glycolysis might be reduced in HFD KO muscle. Most amino acids were elevated in HFD FNDC5 KO muscle. FNDC5 KO raised the levels of liver glucose 6-phosphate and phosphoenolpyruvate, relative to WT, under HFD condition. Glycogen degradation may be reduced in FNDC5 KO livers. FNDC5 KO reduced succinate and increases fumarate under HFD conditions. Liver amino acid levels were elevated in HFD FNDC5 KO livers relative to HFD WT.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eMetabolomic profiling revealed numerous changes to muscle and liver metabolites that suggest broad-ranging effects of FNDC5 on tissue metabolism. The data suggests a number of metabolic pathways to explore further.\u003c/p\u003e","manuscriptTitle":"FNDC5-knock out induces skeletal muscle and liver metabolic disorders in mice, especially in mice with high fat diet","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-04 11:08:35","doi":"10.21203/rs.3.rs-6254178/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"01b20d8b-6654-46a1-b3a9-413e474651d9","owner":[],"postedDate":"April 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46645220,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Diabetes"},{"id":46645221,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Obesity"}],"tags":[],"updatedAt":"2025-05-07T04:38:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-04 11:08:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6254178","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6254178","identity":"rs-6254178","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}