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Detection and Quantitation of Circulating Human Irisin by Tandem Mass Spectrometry
Abstract
Exercise provides many health benefits, including improved metabolism, cardiovascular health, and cognition. We have shown previously that FNDC5, a type I transmembrane protein, and its circulating form, irisin, convey some of these benefits in mice. However, recent reports questioned the existence of circulating human irisin both because human FNDC5 has a non-canonical ATA translation start and because of claims that many human irisin antibodies used in commercial ELISA kits lack required specificity. In this paper we have identified and quantitated human irisin in plasma using mass spectrometry with control peptides enriched with heavy stable isotopes as internal standards. This precise state-of-the-art method shows that human irisin is mainly translated from its non-canonical start codon and circulates at ∼3.6 ng/ml in sedentary individuals; this level is increased to ∼4.3 ng/ml in individuals undergoing aerobic interval training. These data unequivocally demonstrate that human irisin exists, circulates, and is regulated by exercise.
Copyright © 2015 Elsevier Inc. All rights reserved.
... Of note, some studies have shown that FNDC5 mRNA expression in skeletal muscle increased after 12 weeks of training in humans, without significant increase in circulating irisin content [13]. However, many studies in mice and humans have shown that endurance exercise can induce irisin [32][33][34]. Identification and quantification of human irisin in blood by mass spectrometry with stable isotope-enriched control peptides as internal standards have been reported. This precise stateof-the-art method shows that human irisin circulates at a rate of approximately 3.6 ng/ml in sedentary individuals; In individuals undergoing aerobic training, this level increased to approximately 4.3 ng/ml. ...
... This precise stateof-the-art method shows that human irisin circulates at a rate of approximately 3.6 ng/ml in sedentary individuals; In individuals undergoing aerobic training, this level increased to approximately 4.3 ng/ml. These data unequivocally demonstrate that human irisin exists, circulates, and is regulated by exercise [34]. ...
Background and aim
Post-traumatic osteoarthritis (PTOA) is a subtype of osteoarthritis (OA). Exercise may produce and release the myokine irisin through muscle fiber contraction. However, the effect of exercise-promoted irisin production on the internal interactions of the muscle–bone unit in PTOA studies remains unclear.
Methods
Eighteen 8-week-old Sprague–Dawley (SD) rats were randomly divided into three groups: Sham/sedentary (Sham/Sed), PTOA/sedentary (PTOA/Sed), and PTOA/treadmill-walking (PTOA/TW). The PTOA model was established by transection of anterior cruciate ligament (ACLT) and destabilization of medial meniscus (DMM). After 4 weeks of modeling, the PTOA/TW group underwent treadmill exercise (15 m/min, 30 min/d, 5 d/ week, 8 weeks), and the other two groups were free to move in the cage. Evaluation and correlation analysis of muscle, cartilage, subchondral bone and serological indexes were performed after euthanasia.
Results
Eight weeks of treadmill exercise effectively alleviated the trauma-induced OA phenotype, thereby maintaining cartilage and subchondral bone integrity in PTOA, and reducing quadriceps atrophy and myofibril degradation. Exercise reversed the down-regulated expression of peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) and fibronectin type III structural domain protein 5 (FNDC5) in muscle tissue of PTOA rats, and increased the blood irisin level, and the irisin level was positively correlated with the expression of PGC-1α and FNDC5. In addition, correlation analysis showed that irisin metabolism level was strongly negatively correlated with Osteoarthritis Research Society International (OARSI) and subchondral bone loss, indicating that irisin may be involved in cartilage biology and PTOA-related changes in cartilage and subchondral bone. Moreover, the metabolic level of irisin was strongly negatively correlated with muscle fiber cross-sectional area (CSA), Atrogin-1 and muscle ring-finger protein-1(MuRF-1) expression, suggesting that irisin may alleviate muscle atrophy through autocrine action.
Conclusion
Treadmill exercise can alleviate the atrophy and degeneration of muscle fibers in PTOA rats, reduce the degradation of muscle fibrin, promote the expression of serum irisin, and alleviate the degeneration of articular cartilage and subchondral bone loss in PTOA rats. These results indicate that treadmill exercise can affect the process of PTOA by promoting the expression of myokine irisin in rat muscle–bone unit.
... This research clearly shows that human irisin is present, circulates, and is influenced by exercise. (32) Several in vitro experiments, primarily using r-irisin on various cell types, have been published. Different signaling pathways was suggested to be influenced by irisin or involved in the regulation of irisin. ...
BACKGROUND: Fibronectin type III domain-containing protein 5 (FNDC5), or also known as irisin, has been identified for two decades but almost completely disregarded for 10 years. It is present in skeletal muscle, heart, and brain, and in reaction to exercise can transform white adipose tissue into brown. Since then, irisin has gained a lot of attention for its potencies in treating metabolic disorders. In this review article, the potential future of irisin especially on metabolism and aging process will be discussed.CONTENT: Sedentary lifestyle is acknowledged as risk factor for type 2 diabetes mellitus, obesity, immune system issues, asthma, and neurological or heart illness. Irisin is secreted by muscle cells when exercising, produced after the proteolytic cleavage of FNDC5 protein. Irisin has positive impacts on maintaining physiological balance including reducing inflammation, keeping the bone homeostasis, as well as influencing metabolic processes and the neurological system function. Due to these many and advantageous characteristics, irisin could be a possible choice for preventing and managing disorders associated with modern society, and finding the agents to increase irisin can be beneficial.SUMMARY: Irisin offers a fresh potential basis for kinesitherapy and shows promise as a therapeutic target due to its various biological activities. Irisin pathway can be activated through dietary changes, the use of natural substances and drugs and can interact with this signalling pathway which involved peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and uncoupling protein mRNA 1 (UCP1) to resolve obesity and its metabolic comorbidities.KEYWORDS: irisin, FNDC5, exercise, inflammation, obesity, nervous system
... A irisina é um hormônio proteico composto por 112 aminoácidos e é formada, durante o exercício físico, a partir da clivagem da proteína FNDC5, uma proteína precursora transmembrana expressa no músculo. A irisina regula a homeostase da glicose (Lee et al, 2014) e está diretamente relacionada ao aumento do metabolismo energético, à termogênese e à conversão do tecido adiposo branco em tecido adiposo marrom (Lee et al, 2014;Jedrychowski et al, 2015). Níveis maiores de atividade física estão associados com risco reduzido de Doença de Alzheimer e menor declínio cognitivo na doença (Buchman et al, 2012;Choi et al, 2018). ...
Milhões de pessoas sofrem com a Doença de Alzheimer e ainda não há um tratamento-padrão que consiga parar ou reverter a descendente espiral da doença. Este estudo objetivou, com uma revisão narrativa da literatura, conhecer as práticas que vêm sendo utilizadas na prevenção da Doença de Alzheimer entendendo como estas práticas melhoram a qualidade de vida e fornecem opções para uma longevidade mais saudável. Para a busca dos artigos da presente revisão, foi utilizada a base de dados Medline, por meio do PubMed. Os resultados da pesquisa permitiram identificar compostos, como vitaminas D e B12, curcumina, ácido α-lipoico, flavonoides e quercetina, retardando a progressão da Doença de Alzheimer, revertendo alterações inflamatórias no hipocampo, atenuando acúmulo de beta-amiloide ou modulando a resposta imune e se mostrando capazes de melhorar a aprendizagem, a memória e as funções cognitivas em pacientes com Doença de Alzheimer. Diversos estudos demonstram a importância do exercício físico e do sono para o sistema nervoso central e a associação entre distúrbios do sono e distúrbios neurodegenerativos como Doença de Alzheimer, e outros destacam a importância do controle da doença periodontal e dos níveis de açúcar no corpo para a prevenção da Doença de Alzheimer. Com a pesquisa realizada pode-se observar a importância da adoção de estilos de vida saudáveis, da higiene do sono e da boa saúde bucal e intestinal para manter a saúde do cérebro e as faculdades mentais, indicando que pequenas mudanças nos hábitos de cada indivíduo podem impactar o seu futuro.
... By contrast, in a long-term study of 79 subjects with T1DM and 53 normal controls, plasma irisin levels were lower in subjects with T1DM than in controls [47]. Although the reasons for these discrepancies are not known, the method for the detection/quantification of irisin protein may be a critical factor [48], in addition to variables such as age, sex, and physical and treatment status. In our published study [49], we used four complementary methods, including western blot, immunohistochemistry, protein liquid chip assay, and liquid chromatographytandem mass spectrometry (LC-MS), to quantify irisin in lung epithelial cells and tissues. ...
Background
Diabetic cardiomyopathy (DCM) is a serious complication in patients with type 1 diabetes mellitus (T1DM), which still lacks adequate therapy. Irisin, a cleavage peptide off fibronectin type III domain-containing 5, has been shown to preserve cardiac function in cardiac ischemia–reperfusion injury. Whether or not irisin plays a cardioprotective role in DCM is not known.
Methods and results
T1DM was induced by multiple low-dose intraperitoneal injections of streptozotocin (STZ). Our current study showed that irisin expression/level was lower in the heart and serum of mice with STZ-induced TIDM. Irisin supplementation by intraperitoneal injection improved the impaired cardiac function in mice with DCM, which was ascribed to the inhibition of ferroptosis, because the increased ferroptosis, associated with increased cardiac malondialdehyde (MDA), decreased reduced glutathione (GSH) and protein expressions of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), was ameliorated by irisin. In the presence of erastin, a ferroptosis inducer, the irisin-mediated protective effects were blocked. Mechanistically, irisin treatment increased Sirtuin 1 (SIRT1) and decreased p53 K382 acetylation, which decreased p53 protein expression by increasing its degradation, consequently upregulated SLC7A11 and GPX4 expressions. Thus, irisin-mediated reduction in p53 decreases ferroptosis and protects cardiomyocytes against injury due to high glucose.
Conclusion
This study demonstrated that irisin could improve cardiac function by suppressing ferroptosis in T1DM via the SIRT1-p53-SLC7A11/GPX4 pathway. Irisin may be a therapeutic approach in the management of T1DM-induced cardiomyopathy.
... Moreover, different commercial ELISA kits have been reported to detect irisin levels in a range from picograms to micrograms per milliliter of serum or plasma [14,15]. In response, Jedrychowski et al. performed a precise quantitative assay of human irisin in plasma using mass spectrometry, providing substantial progress in the detection of this peptide [16]. Mass spectrometry is considered the gold standard, but only a few studies have employed this method [14]. ...
Recently discovered irisin, a member of the myokines family, is a potential mediator of exercise-induced energy metabolism and a factor promoting browning of the white adipose tissue. Recent evidence indicates that this myokine, released from contracting muscles, can mediate the beneficial effects of exercise on health. Irisin may be a potential therapeutic agent against obesity and has been shown to play an important role in the protection of various cells, tissues, and organs due to its anti-inflammatory, antioxidative, and anti-cancer properties. Our aim was to review the recent experimental and clinical studies on irisin and its expression, release into the bloodstream, tissue targets, and potential contribution to the protective effects of exercise in the gastrointestinal tract. Particular emphasis was placed on inflammatory bowel disease, intestinal ischemia/reperfusion injury, periodontitis, and other digestive tract disorders, including carcinogenesis. Overall, irisin holds significant potential as a novel target molecule, offering a safe and therapeutic approach to treating various gastrointestinal diseases.
... While the ATA codon in human FNDC5 may represent a null mutation, humans were presumed incapable of producing irisin (7,8). The key findings using modified mass spectrometry techniques confirmed that irisin was real and expressed mainly at the atypical ATA start codon of FNDC5 (9). Subsequently, irisin has been shown to bind to its receptor aV/b5 integrin, thereby playing a protective role in bone remodeling and gut barrier function (10)(11)(12). ...
Vascular calcification is a common accompanying pathological change in many chronic diseases, which is caused by calcium deposition in the blood vessel wall and leads to abnormal blood vessel function. With the progress of medical technology, the diagnosis rate of vascular calcification has explosively increased. However, due to its mechanism’s complexity, no effective drug can relieve or even reverse vascular calcification. Irisin is a myogenic cytokine regulating adipose tissue browning, energy metabolism, glucose metabolism, and other physiological processes. Previous studies have shown that irisin could serve as a predictor for vascular calcification, and protect against hypertension, diabetes, chronic kidney disease, and other risk factors for vascular calcification. In terms of mechanism, it improves vascular endothelial dysfunction and phenotypic transformation of vascular smooth muscle cells. All the above evidence suggests that irisin plays a predictive and protective role in vascular calcification. In this review, we summarize the association of irisin to the related risk factors for vascular calcification and mainly explore the role of irisin in vascular calcification.
... This, in turn, enhances the expression of a protein called FNDC5, leading to the release of more irisin into the bloodstream through the cleavage of FNDC5. The level of irisin in the plasma is elevated in individuals who engage in aerobic training [86]. Specifically, exercise stimulates the production of FNDC5 and BDNF in the hippocampus. ...
Addressing cognitive impairment (CI) represents a significant global challenge in health and social care. Evidence suggests that aging and metabolic disorders increase the risk of CI, yet promisingly, physical exercise has been identified as a potential ameliorative factor. Specifically, there is a growing understanding that exercise-induced cognitive improvement may be mediated by molecules known as exerkines. This review delves into the potential impact of aging and metabolic disorders on CI, elucidating the mechanisms through which various exerkines may bolster cognitive function in this context. Additionally, the discussion extends to the role of exerkines in facilitating stem cell mobilization, offering a potential avenue for improving cognitive impairment.
... It was discovered that irisin, a myokine released into the bloodstream during physical activity, can induce thermogenesis and adipocyte browning in both humans and mice [9]. The transmembrane precursor protein fibronectin type III domain containing 5 (FNDC5), which is expressed in muscle and regulated by peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), is cleaved from irisin. ...
... When Spiegelman et al. discovered irisin and its biological properties [56], an attempt was made to determine the physiological range of its concentrations during the resting state and after physical activity. Jędrychowski et al. showed an increase in the concentration of irisin in physically active persons in comparison with people who led a sedentary lifestyle (4.3 ng/mL vs. 3.6 ng/mL; p < 0.05) [57]. Moreno et al. also showed that the concentration of irisin is higher for people who are physically active than in those who lead a sedentary lifestyle (128.55 µg/mL ± 78.71 µg/mL vs. 105.66 ...
Simple Summary
Cachexia and malnutrition are characteristic of oncology patients, including those with head and neck cancer (HNSCC). Fifty-six patients diagnosed with HNSCC (study group) were included in the study, and seventy patients constituted the control group. We used standardized questionnaires to assess nutritional status and cachexia. In our study, we evaluated whether ghrelin, titin, and irisin can be a useful diagnostic marker, a prognostic marker of cancer development in these patients, and whether the levels of these proteins depend on the nutritional status of the patients. We used molecular biology methods to evaluate the concentration of the selected proteins. The results indicate that the levels of ghrelin, titin, and irisin correlate well with the nutritional and cachexia status of patients with HNSCC. Until now, to our best knowledge, this is the first study assessing the changes in concentrations of these proteins in this type of cancer.
Abstract
The goal of this paper was the evaluation of the changes in the expression profile of irisin, ghrelin, and titin in the carcinoma tissue and in the blood of patients with head and neck squamous cell carcinoma (HNSCC), including determining the profile of their expression in relation to patient nutrition. The study included 56 patients with diagnosed squamous cell carcinoma of HNSCC in the T3 and T4 stages of the disease. Healthy control tissue specimens were collected from an area 10 mm outside the histologically negative margin. In turn, the blood and serum from the control group came from healthy volunteers treated for non-oncologic reasons (n = 70). The molecular analysis allowed us to determine the profile of irisin, ghrelin, and titin methylation, evaluate their expression on the level of mRNA (quantitative Reverse Transcription Polymerase Chain Reaction; qRT-PCR) and protein (Enzyme-Linked Immunosorbent Assay Reaction; ELISA) in the carcinoma tissue and the margin of healthy tissue, as well as in serum of patients in the study and control groups. At the start of our observations, a Body Mass Index (BMI) < 18.5 was noted in 42 of the patients, while six months after the treatment a BMI < 18.5 was noted in 29 patients. We also noted a decrease in the expression of irisin, ghrelin, and titin both on the level of mRNA and protein, as well as a potential regulation of their expression via DNA methylation. There is no convincing evidence that the proteins assayed in the present work are specific with regard to HNSSC.
... Irisin is an exfoliated extracellular domain of the transmembrane protein FNDC5, which is secreted from skeletal muscle and other organs and increases with exercise (Huh et al., 2012;Jedrychowski et al., 2015). Exercise enhances the activity and expression of some receptors, such as peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) (Raefsky and Mattson, 2017). ...
Background
This study was designed to investigate the relationship of irisin with the severity of Parkinson’s disease (PD) and dopamine (DOPA) uptake in patients with PD and to understand the role of irisin in PD.
Methods
The plasma levels of irisin and α-syn were measured by enzyme-linked immunosorbent assay (ELISA). Motor and nonmotor symptoms were assessed with the relevant scales. DOPA uptake was measured with DOPA positron emission tomography (PET)/magnetic resonance imaging (MRI).
Results
The plasma levels of α-syn and irisin in patients with PD gradually increased and decreased, respectively, with the progression of the disease. There was a negative correlation between plasma α-syn and irisin levels in patients with PD. The level of irisin in plasma was negatively correlated with Unified Parkinson’s Disease Rating Scale (UPDRS)-III scores and positively correlated with Montreal Cognitive Assessment (MoCA) scores. The striatal/occipital lobe uptake ratios (SORs) of the ipsilateral and contralateral caudate nucleus and anterior and posterior putamen in the high-irisin group were significantly higher than those in the low-irisin group, and irisin levels in the caudate nucleus and anterior and posterior putamen contralateral to the affected limb were lower than those on the ipsilateral side. The level of irisin was positively correlated with the SORs of the ipsilateral and contralateral caudate nucleus and putamen in PD patients.
Conclusions
Irisin plays a neuroprotective role by decreasing the level of α-syn. Irisin is negatively correlated with the severity of motor symptoms and cognitive impairment. More importantly, irisin can improve DOPA uptake in the striatum of patients with PD, especially on the side contralateral to the affected limb.
... Irisin is a myokine formed by the cleavage of fibronectin type III domain containing 5 proteins (FNDC5) by proteins of the disintegrin and metallopeptidase (ADAM) family, such as ADAM10, to form the 112 aa peptide irisin [95][96][97]. Most likely, irisin combines with integrin αVβ1 in adipocytes [98,99]. ...
Obesity is a characteristic disease of the twenty-first century that is affecting an increasing percentage of society. Obesity expresses itself in different phenotypes: normal-weight obesity (NWO), metabolically obese normal-weight (MONW), metabolically healthy obesity (MHO), and metabolically unhealthy obesity (MUO). A range of pathophysiological mechanisms underlie the occurrence of obesity, including inflammation, oxidative stress, adipokine secretion, and other processes related to the pathophysiology of adipose tissue (AT). Body mass index (BMI) is the key indicator in the diagnosis of obesity; however, in the case of the NWO and MONW phenotypes, the metabolic disturbances are present despite BMI being within the normal range. On the other hand, MHO subjects with elevated BMI values do not present metabolic abnormalities. The MUO phenotype involves both a high BMI value and an abnormal metabolic profile. In this regard, attention has been focused on the variety of molecules produced by AT and their role in the development of obesity. Nesfatin-1, neuregulin 4, myonectin, irisin, and brain-derived neurotrophic factor (BDNF) all seem to have protective effects against obesity. The primary mechanism underlying the action of nesfatin-1 involves an increase in insulin sensitivity and reduced food intake. Neuregulin 4 sup-presses lipogenesis, decreases lipid accumulation, and reduces chronic low-grade inflammation. Myonectin lowers the amount of fatty acids in the bloodstream by increasing their absorption in the liver and AT. Irisin stimulates the browning of white adipose tissue (WAT) and consequently in-creases energy expenditure, additionally regulating glucose metabolism. Another molecule, BDNF, has anorexigenic effects. Decorin protects against the development of hyperglycemia, but may also contribute to proinflammatory processes. Similar effects are shown in the case of visfatin and chemerin, which may predispose to obesity. Visfatin increases adipogenesis, causes cholesterol accumulation in macrophages, and contributes to the development of glucose intolerance. Chemerin induces angiogenesis, which promotes the expansion of AT. This review aims to discuss the role of adipokines and myokines in the pathogenesis of the different obesity phenotypes.
... 1,2 Irisin was initially reported to reprogramme adipocyte metabolism and control peripheral glucose homeostasis. 3,4 FNDC5 single nucleotide polymorphisms (SNPs) have been associated with obesity and metabolic syndrome in humans. [5][6][7] In the brain, FNDC5/irisin induces the expression of neurotrophins and synaptic plasticity-related genes 2,8 and mediates, at least in part, exercise-induced neurogenesis in the mouse hippocampus. ...
Fibronectin type III domain–containing protein 5 (FNDC5) and its derived hormone, irisin, have been associated with metabolic control in humans, with described FNDC5 single nucleotide polymorphisms being linked to obesity and metabolic syndrome. Decreased brain FNDC5/irisin has been reported in subjects with dementia due to Alzheimer’s disease. Since impaired brain glucose metabolism develops in ageing and is prominent in Alzheimer’s disease, here, we examined associations of a single nucleotide polymorphism in the FNDC5 gene (rs1746661) with brain glucose metabolism and amyloid-β deposition in a cohort of 240 cognitively unimpaired and 485 cognitively impaired elderly individuals from the Alzheimer’s Disease Neuroimaging Initiative. In cognitively unimpaired elderly individuals harbouring the FNDC5 rs1746661(T) allele, we observed a regional reduction in low glucose metabolism in memory-linked brain regions and increased brain amyloid-β PET load. No differences in cognition or levels of cerebrospinal fluid amyloid-β42, phosphorylated tau and total tau were observed between FNDC5 rs1746661(T) allele carriers and non-carriers. Our results indicate that a genetic variant of FNDC5 is associated with low brain glucose metabolism in elderly individuals and suggest that FNDC5 may participate in the regulation of brain metabolism in brain regions vulnerable to Alzheimer’s disease pathophysiology. Understanding the associations between genetic variants in metabolism-linked genes and metabolic brain signatures may contribute to elucidating genetic modulators of brain metabolism in humans.
... 1B). To confirm the R 21 /A mutation at the protein level we developed a novel in-gel digestion-based mass spectrometry (MS) analysis for TLQP-21 using an approach similar to what was used for other peptides [28] ( Figure 3A). Fresh adrenal gland was rapidly lysed in RIPA lysis buffer and equal amounts of proteins were loaded in a 12% precast gel and were resolved in the MES buffer system along with the synthetic TLQP-21 peptide. ...
Objective:
Pro-peptide precursors are processed into biologically active peptide hormones or neurotransmitters, each playing an essential role in physiology and disease. Genetic loss of function of a pro-peptide precursor results in the simultaneous ablation of all biologically-active peptides within that precursor, often leading to a composite phenotype that can be difficult to align with the loss of specific peptide components. Due to this biological constraint and technical limitations, mice carrying the selective ablation of individual peptides encoded by pro-peptide precursor genes, while leaving the other peptides unaffected, have remained largely unaddressed.
Methods:
We developed and characterized a mouse model carrying the selective knockout of the TLQP-21 neuropeptide (ΔTLQP-21) encoded by the Vgf gene. To achieve this goal, we used a knowledge-based approach by mutating a codon in the Vgf sequence leading to the substitution of the C-terminal Arginine of TLQP-21, which is the pharmacophore as well as an essential cleavage site from its precursor, into Alanine (R21→A).
Results:
We provide several independent validations of this mouse, including a novel in-gel digestion targeted mass spectrometry identification of the unnatural mutant sequence, exclusive to the mutant mouse. ΔTLQP-21 mice do not manifest gross behavioral and metabolic abnormalities and reproduce well, yet they have a unique metabolic phenotype characterized by an environmental temperature-dependent resistance to diet-induced obesity and activation of the brown adipose tissue.
Conclusions:
The ΔTLQP-21 mouse line can be a valuable resource to conduct mechanistic studies on the necessary role of TLQP-21 in physiology and disease, while also serving as a platform to test the specificity of novel antibodies or immunoassays directed at TLQP-21. Our approach also has far-reaching implications by informing the development of knowledge-based genetic engineering approaches to generate selective loss of function of other peptides encoded by pro-hormones genes, leaving all other peptides within the pro-protein precursor intact and unmodified.
... Exercise-induced activation of PGC-1α can induce skeletal muscle to secrete FNDC5/Irisin [88]. Plasma Irisin levels in people who exercise regularly are significantly higher than those who lead sedentary lifestyles [89]. Wrann et al. [82] found that exercise can increase the expression of FNDC5 and BDNF in skeletal muscle and hippocampal tissue. ...
Depression is a common mental disorder in which patients often experience feelings of sadness, fatigue, loss of interest, and pleasure. Exercise is a widely used intervention for managing depression, but the specific molecular mechanisms underlying its antidepressant effect are unclear. In this narrative review, we aim to synthesize current knowledge on the molecular, neural, and physiological mechanisms through which exercise exerts its antidepressant effect and discuss the various exercise interventions used for managing depression. We conducted a narrative review of the literature on the topic of exercise and depression. Our review suggests that exercise impacts peripheral tryptophan metabolism, central inflammation, and brain-derived neurotrophic factors through the peroxisome proliferator-activated receptor γ activating factor 1α (PGC-1α) in skeletal muscles. The uncarboxylated osteocalcin facilitates “bone-brain crosstalk”, and exercise corrects atypical expression of brain-gut peptides, modulates cytokine production and neurotransmitter release, and regulates inflammatory pathways and microRNA expression. Aerobic exercise is recommended at frequencies of 3 to 5 times per week with medium to high intensity. Here we highlight the significant potential of exercise therapy in managing depression, supported by the molecular, neural, and physiological mechanisms underlying its antidepressant effect. Understanding the molecular pathways and neural mechanisms involved in exercise’s antidepressant effect opens new avenues for developing novel therapies for managing depression.
... Furthermore, the quality of commercially available ELISA kits for circulating irisin was skepticized. Moreover, it was suggested that the validation of the irisin values toward tandem mass spectrometry was a better option to validate the irisin values toward tandem mass spectrometry [63]. ...
Background:
Adipokines are considered to be involved in the pathogenesis of diabetes and depression. The associations of serum levels of leptin and irisin with depressive symptoms were investigated in elderly patients with type 2 diabetes (T2DM).
Methods:
189 elderly diabetics were assessed with the 30-item Geriatric Depression Scale (GDS-30), and 57 patients with depressive symptoms and 132 controls were selected. Blood biochemical parameters, including serum irisin and leptin, were measured.
Results:
Serum irisin levels were decreased and leptin concentrations were significantly higher in T2DM patients with depressive symptoms compared to controls. In all subjects, the irisin level was inversely correlated with the leptin level and the GDS-30 score, whereas the leptin level was highly correlated with BMI and the GDS-30 score. Higher levels of leptin and lower concentrations of irisin are, among other factors, variables indicative of predictive capacity for depressive symptoms in elderly patients with T2DM.
Conclusions:
The results indicated that irisin and leptin levels may be used as diagnostic markers of depressive symptoms in diabetic, elderly patients and as potential therapeutic targets for the treatment. Further prospective and more extensive studies are needed to clarify the role of these adipokines in the common pathogenesis of depression and diabetes.
... Irisin is an exercise-induced myokine that modulates adipose, bone and brain functions (Boström et al., 2012;Kim et al., 2018;De Freitas et al., 2020;Isaac et al., 2021;Jodeiri Farshbaf and Alvina, 2021). Upon exercise, irisin is cleaved from a precursor protein, fibronectin type III domain containing protein 5 (FNDC5), and released into the circulation (Boström et al., 2012;Jedrychowski et al., 2015). FNDC5 and irisin have been detected in the brain (Wrann et al., 2013;Lourenco et al., 2019), and irisin has been shown to mediate the beneficial actions of physical exercise in mouse models of Alzheimer's disease (AD) (Lourenco et al., 2019). ...
Background
Irisin is an exercise‐linked myokine produced by cleavage of the membrane precursor fibronectin type III domain‐containing protein 5 (FNDC5) in skeletal muscle, brain, and other tissues. Recently, irisin has been identified as a key molecule for exercise‐induced neuroprotection in mouse models of Alzheimer’s disease (AD). However, the role of FNDC5/irisin in AD pathology has not yet been comprehensively explored. Here, we investigated potential mechanisms underlying neuroprotection induced by FNDC5/irisin in AD.
Method
Primary rat hippocampal neuronal cultures were transduced with adenoviral vectors to express FNDC5 (AdFNDC5) or GFP (as a control) or exposed to recombinant irisin (25 nM) for defined timepoints. Brain‐derived neurotrophic factor (BDNF) levels were measured by ELISA; extracellular signal‐regulated kinase 1/2 (ERK 1/2) phosphorylation was determined by Western blotting; and the effects of irisin on amyloid‐β oligomers (AβO)‐induced reactive oxygen species (ROS) accumulation was determined by the DCF fluorescence. Finally, we analyzed RNA‐seq data from human postmortem hippocampi within The Aging, Dementia and Traumatic Brain Injury Study to determine potential correlations between FNDC5 gene expression and AD pathology.
Result
We found that irisin stimulates extracellular BDNF accumulation, ERK 1/2 phosphorylation ( i.e . activation) and prevents AβOs‐induced ROS accumulation in primary hippocampal neurons. Analysis of RNA‐seq data indicates that hippocampal FNDC5 gene expression is reduced with aging and tau (assessed by Braak neuropathological scale and hippocampal phospho‐tau immunoreactivity), but not with amyloid pathology in humans.
Conclusion
These results indicate potential mechanisms by which FNDC5/irisin signaling promotes neuroprotection and support the notion that stimulation of irisin signaling may be beneficial against neurodegeneration in AD.
... Fibronectin Type III Domain-Containing Protein 5/ Irisin, Brain-Derived Neurotrophic Factor, and Brain AMPK Gene arrays of muscle from AMPKβ1β2 muscle-specific null mice identified that 1 of the top downregulated genes was Fndc5 (fibronectin type III domain containing 5) (259). Fndc5, the cell membrane bound protein precursor of irisin, was first characterized as a PGC1α regulated gene product which encodes irisin, which is the cleaved from fibronectin type III domain-containing protein 5 (FNDC5) that enters circulation, although it should be noted there is some concern that irisin may not circulate in humans (260)(261)(262). Irisin was first described as an exercise-induced myokine that promoted adipose tissue browning in mice, an effect that was later shown to be dependent on AMPK (263,264). ...
Complex multi-cellular organisms require a coordinated response from multiple tissues to maintain whole-body homeostasis in the face of energetic stressors like fasting, cold and exercise. It is also essential that energy is stored efficiently with feeding and the chronic nutrient surplus that occurs with obesity. Mammals have adapted several endocrine signals that regulate metabolism in response to changes in nutrient availability and energy demand. These include hormones altered by fasting and refeeding including insulin, glucagon, GLP-1 (glucagon like peptide-1), catecholamines, ghrelin and FGF21 (fibroblast growth factor 21); adipokines such as leptin and adiponectin; cell stress-induced cytokines like TNFα (tumor necrosis factor alpha) and GDF15 (growth differentiating factor 15), and lastly exerkines such as IL-6 (interleukin-6) and irisin. Over the last two decades, it has become apparent that many of these endocrine factors control metabolism by regulating the activity of the AMPK (AMP-activated protein kinase). AMPK is a master regulator of nutrient homeostasis, phosphorylating over 100-distinct substrates that are critical for controlling autophagy, carbohydrate, fatty acid, cholesterol and protein metabolism. In this review we discuss how AMPK integrates endocrine signals to maintain energy balance in response to diverse homeostatic challenges. We also present some considerations with respect to experimental design which should enhance reproducibility and the fidelity of the conclusions.
... 108 The current gold-standard irisin measurement by tandem mass spectrometry is not yet available for wide scale use. 109 Insulin-like growth factor-I (IGF-I) and Fibroblast Growth Factor-2 (FGF-2) are well known osteogenic factors. 110 IGF-I is one of the more ubiquitous and pleiotropic hormones acting in endocrine, paracrine, and autocrine actions and has been associated with a number of health and fitness outcomes. ...
Introduction:
Bone stress injuries (BSIs) have plagued the military for over 150 years; they afflict around 5 to 10% of military recruits, more so in women, and continue to place a medical and financial burden on defence. While the tibia generally adapts to the rigours of basic military training, the putative mechanisms for bone maladaptation are still unclear.
Methods:
This paper provides a review of the published literature on current risk factors and emerging biomarkers for BSIs in military personnel; the potential for biochemical markers of bone metabolism to monitor the response to military training; and, the association of novel biochemical 'exerkines' with bone health.
Results:
The primary risk factor for BSI in military (and athletic) populations is too much training, too soon. Appropriate physical preparation before training will likely be most protective, but routine biomarkers will not yet identify those at risk. Nutritional interventions will support a bone anabolic response to training, but exposure to stress, sleep loss, and medication is likely harmful to bone. Monitoring physiology using wearables-ovulation, sleep and stress-offer potential to inform prevention strategies.
Conclusions:
The risk factors for BSIs are well described, but their aetiology is very complex particularly in the multi-stressor military environment. Our understanding of the skeletal responses to military training is improving as technology advances, and potential biomarkers are constantly emerging, but sophisticated and integrated approaches to prevention of BSI are warranted.
... Although immunohistochemical studies have shown that irisin is found in the pancreas, liver, and stomach, it is mainly secreted from skeletal muscle [3] . Following acute bouts of exercise, FDNC5 expression is affected by regulation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), a multispecific coactivator of transcription, which is responsible for multiple gene regulation in response to nutritional and physiological signals in tissues, and subsequently cleaved to irisin, released into the circulation [4] . ...
Purpose: Irisin is a hormone-like molecule secreted from skeletal muscle in response to exercise both in mice and in humans and identified as an important effector in the crosstalk between muscle and bone. Although a number of studies report that irisin increased osteoblast differentiation in vitro and cortical bone mass in vivo, the models used are exclusively rodent ones. Due to the lack of reports on human cell models, the aim of our work was to investigate the in vitro effects of irisin on the proliferation and the osteogenic differentiation processes in human adipose tissue-derived mesenchymal stem cells (hAMSCs). Methods: hAMSCs were obtained by enzymatic digestion and mechanical dispersion, and cultured in growth medium. Cells were exposed to 10 and 100 ng/ml irisin for the entire experimental period and refreshed every two days. The proliferation was performed in growth medium containing 2.5% fetal bovine serum, and measured by cell counting at 24-48-72 hours. Alkaline phosphatase (ALP) activity and Ca2+ depositions were quantified by fluorometric assay during up to 35 days of osteogenic induction. Results: Cell proliferation assay showed that 100 ng/ml irisin significantly increased the proliferation process (p<0.01) vs control, with a decrease of cell doubling time from 88 to 63 hours. Osteodifferentiation with 10 and 100 ng/ml irisin showed significant increases in ALP activity vs control (p<0.01) after 14 days. Moreover, both tested concentrations of irisin were able to accelerate the deposition of mineralized matrix, resulting in significant increments in the production of Ca2+ nodules vs control after 35 days (p<0.01). Conclusions: This work showed the in vitro effects of irisin on a human cell model of AMSCs. The preliminary results show this myokine to be an important effector on cell proliferation and during osteo-differentiation of hAMSCs, supporting the hypothesis that irisin could represent a potent new anabolic treatment to bring about gain of bone mass.
... Since the first discovery of irisin, scientists have tried to understand and determine the cleavage protein for FNDC5 (46). In fact, by using an improved mass spectrometry technique with synthetic peptides rich in heavy stable isotopes as internal standards, Jedrychowski et al. found that irisin is mainly expressed in the non-canonical start codon of FNDC5 (47). Based on recent studies, irisin binds to proteins of the aV class of integrins. ...
Human beings lead largely sedentary lives. From an evolutionary perspective, such lifestyle is not beneficial to health. Exercise can promote many enabling pathways, particularly through circulating exerkines, to optimize individual health and quality of life. Such benefits might explain the protective effects of exercise against aging and noncommunicable diseases. Nevertheless, the miRNA-mediated molecular mechanisms and exerkine interorgan crosstalk that underlie the beneficial effects of exercise remain poorly understood. In this mini review, we focused on the exerkine, irisin, mainly produced by muscle contraction during adaptation to exercise and its beneficial effects on body homeostasis. Herein, the complex role of irisin in metabolism and inflammation is described, including its subsequent effects on thermogenesis through browning to control obesity and improve glycemic regulation for diabetes mellitus control, its potential to improve cognitive function (via brain derived neurotrophic factor), and its pathways of action and role in aging.
Neuroplasticity is an integral feature of both the developing brain and the brain maintaining functional homeostasis and implementing adaptive changes at normal conditions and upon compensation for pathology. Support of neuroplasticity mechanisms of is one of the targets for therapeutic intervention in the treatment of neurodegenerative and stress-associated diseases. Progress in understanding the mechanisms of interaction between the muscular system and the brain points to the role of the myokine irisin in mediating the procognitive and antidepressant activity of physical exercises. Irisin being released upon myocytes activation in the periphery can cross the blood-brain barrier and is thought to stimulate cellular autophagy. Autophagy-mediated activation of protein and macromolecule recycling promotes adaptive restructuring of synaptic contacts, and the release of proteases, including matrix metalloproteinase 9, which are determining the reformatting of the extracellular matrix, maturation of brain-derived neurotrophic factor (BDNF), and, therefore, the positive regulation of BDNF signaling. Recent findings allow one to consider factors stimulating autophagy as prerequisites for successful treatment of neurological and psychiatric disorders, as well as age-related dementia. Therefore, irisin, as a physiological regulator of autophagy, appears as a prototype molecule for the creation of new therapeutic agents for the correction of neurodegenerative conditions and stress-associated brain disorders.
The present study was conducted to investigate the effect of eight weeks of aerobic training and vitamin D3 supplementation on cardiac irisin protein levels, insulin resistance and lipid profile in rats induced with type 2 diabetes.
Cardiovascular disease (CVD) is an important factor threatening the health of the elderly. Aging leads to changes in the structure and function of the cardiovascular system, which increases the risk of CVD in the elderly. Cardiac aging is characterized by increased left ventricular wall thickness, increased degree of myocardial fibrosis, increased cardiac hardness, and decreased cardiac function, while vascular aging is characterized by enlarged lumen, thickened wall, and endothelial dysfunction. Promoting healthy cardiovascular aging means reducing the age-related cardiovascular dysfunction and the risks of CVD. Exercise is a crucial means for the treatment and rehabilitation of CVD. Exercise reduces the risk factors of CVD, remodels the cardiovascular structure, and increases the resistance of heart to detrimental stimulus, which promotes healthy cardiovascular aging. The improved mitochondrial function via exercise plays a key role in the health effects of exercise. In addition, exercise promotes the secretion of exerkines in various tissues and organs, which plays a role in reducing inflammation, improving metabolism, inhibiting apoptosis, etc., thus benefiting cardiovascular health. This review discusses the mechanism and potential application of exercise in promoting healthy cardiovascular aging. Exploring the specific mechanisms underlying exercise-induced cardiovascular health and formulating accurate exercise prescriptions for different populations is an important direction to promote healthy cardiovascular aging and prevent CVD.
The purpose of this narrative review was to investigate the key determinants of musculoskeletal health in childhood and adolescence, with particular attention to the role of physical activity. First, we examined the importance of bone modeling and remodeling in maintaining the bone health and the integrity and mechanical characteristic of the skeleton. In addition, we reported the evidence on an appropriate calcium and vitamin D intake, as well as local load variation in achieving proper peak bone mass. Proteomic and transcriptomic studies identified the skeletal muscle “secretoma”, consisting of several myokines involved in endocrine and paracrine functions. Among these, we explored the role of irisin, a myokine involved in the muscle-bone crosstalk, and in the regulation of metabolic pathways. It is known that physical activity during growing positively impacts on skeleton and can protect by bone loss in adulthood. However, there are still concerns about the optimal interval duration and exercise intensity, particularly at the pubertal growth spurt which represents a window of opportunity to increase skeletal strength. We reported data from clinical trials performed in the last 5 years analyzing the impact of the type and timing of physical activity during childhood on skeletal development. Finally, we reported recent data on the significance of physical activity in some rare diseases.
Mammalian FNDC5 encodes a protein precursor of Irisin, which is important for exercise-dependent regulation of whole-body metabolism. In a genetic screen in Drosophila , we identified Iditarod ( Idit ), which shows substantial protein homology to mouse and human FNDC5 , as a regulator of autophagy acting downstream of Atg1/Atg13. Physiologically, Idit -deficient flies showed reduced exercise performance and defective cold resistance, which were rescued by exogenous expression of Idit . Exercise training increased endurance in wild-type flies, but not in Idit -deficient flies. Conversely, Idit is induced upon exercise training, and transgenic expression of Idit in wild-type flies increased endurance to the level of exercise trained flies. Finally, Idit deficiency prevented both exercise-induced increase in cardiac Atg8 and exercise-induced cardiac stress resistance, suggesting that cardiac autophagy may be an additional mechanism by which Idit is involved in the adaptive response to exercise. Our work suggests an ancient role of an Iditarod/Irisin/FNDC5 family of proteins in autophagy, exercise physiology, and cold adaptation, conserved throughout metazoan species.
A pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid-β (Aβ) protein in the brain. Physical exercise has been shown to reduce Aβ burden in various AD mouse models, but the underlying mechanisms have not been elucidated. Irisin, an exercise-induced hormone, is the secreted form of fibronectin type-III-domain-containing 5 (FNDC5). Here, using a three-dimensional (3D) cell culture model of AD, we show that irisin significantly reduces Aβ pathology by increasing astrocytic release of the Aβ-degrading enzyme neprilysin (NEP). This is mediated by downregulation of ERK-STAT3 signaling. Finally, we show that integrin αV/β5 acts as the irisin receptor on astrocytes required for irisin-induced release of astrocytic NEP, leading to clearance of Aβ. Our findings reveal for the first time a cellular and molecular mechanism by which exercise-induced irisin attenuates Aβ pathology, suggesting a new target pathway for therapies aimed at the prevention and treatment of AD.
Androgens' pleiotropic actions in promoting sex differences present not only a challenge to providing a comprehensive account of their function, but also an opportunity to gain insights by comparing androgenic actions across organ systems. Although often overlooked by neuroscientists, skeletal muscle is another androgen-responsive organ system which shares with the nervous system properties of electrochemical excitability, behavioral relevance, and remarkable capacity for adaptive plasticity. Here we review androgenic regulation of mitogenic plasticity in skeletal muscle with the goal of identifying areas of interest to those researching androgenic mechanisms mediating sexual differentiation of neurogenesis. We use an organizational-activational framework to relate broad areas of similarity and difference between androgen effects on mitogenesis in muscle and brain throughout the lifespan, from early organogenesis, through pubertal organization, adult activation, and aging. The focus of the review is androgenic regulation of muscle-specific stem cells (satellite cells), which share with neural stem cells essential functions in development, plasticity, and repair, albeit with distinct, muscle-specific features. Also considered are areas of paracrine and endocrine interaction between androgen action on muscle and nervous system, including mediation of neural plasticity of innervating and distal neural populations by muscle-produced trophic factors.
Digestive system diseases (DSD) are very complex conditions that severely threaten human health. Therefore, there is an urgent need to develop new pharmacological treatment strategies. Irisin, a myokine discovered in 2012, is produced by fibronectin type III domain-containing protein 5 (FNDC5), which is a transmembrane protein. Irisin is involved in promoting the browning of white adipose tissue, the regulation of energy metabolism, and the improvement of insulin resistance. Irisin is also an essential mediator of the inflammatory response, oxidative stress, and cell apoptosis. Recent studies have proved that irisin concentration is altered in DSD and exerts pivotal effects on the initiation, progression, and prognosis of these diseases through various mechanisms. Therefore, studying the expression and function of irisin may have great significance for the diagnosis and treatment of DSD. Here, we focus on irisin and explore the multiple molecular pathways targeted by irisin therapy. This review indicates that irisin can serve as a diagnostic marker or potential therapeutic agent for DSD. DATA AVAILABILITY: Not applicable.
Introduction:
Physical activity benefits patients with Parkinson's disease (PD) and is assumed to possess disease-modifying potential. PD-related biomarkers, such as dopamine transporter (DAT) imaging and cerebrospinal fluid (CSF) α-synuclein (α-syn) and amyloid β (Aβ), correlate with disease severity and, to some extent, reflect disease progression and pathology. However, the association between regular physical activity and PD biomarker changes remains unknown. This study aimed to investigate the association between physical activity and longitudinal trajectories of PD biomarkers.
Methods:
This retrospective study included 444 patients with a median follow-up time of 5 years from the Parkinson's Progression Markers Initiative cohort. Data collection included physical activity as scaled by the Physical Activity Scale for the Elderly questionnaire, dopamine transporter imaging, CSF assessment, and serum biomarkers. We analyzed the data using a linear mixed regression model.
Results:
Regular physical activity was associated with a slower decline of DAT uptake in the caudate (β = 0.063, p = 0.011) and the putamen (β = 0.062, p = 0.023). No association was detected between regular physical activity and CSF, as well as serum biomarkers.
Conclusion:
Regular physical activity is associated with favorable PD biomarker progression, indicating a potential disease-modifying effect.
Introduction:
Physical activity benefits patients with Parkinson's disease (PD) and is assumed to possess disease-modifying potential. PD-related biomarkers, such as dopamine transporter (DAT) imaging and cerebrospinal fluid (CSF) α-synuclein (α-syn) and amyloid β (Aβ), correlate with disease severity and, to some extent, reflect disease progression and pathology. However, the association between regular physical activity and PD biomarker changes remains unknown. This study aimed to investigate the association between physical activity and longitudinal trajectories of PD biomarkers.
Methods:
This retrospective study included 444 patients with a median follow-up time of 5 years from the Parkinson's Progression Markers Initiative cohort. Data collection included physical activity as scaled by the Physical Activity Scale for the Elderly questionnaire, dopamine transporter imaging, CSF assessment, and serum biomarkers. We analyzed the data using a linear mixed regression model.
Results:
Regular physical activity was associated with a slower decline of DAT uptake in the caudate (β = 0.063, p = 0.011) and the putamen (β = 0.062, p = 0.023). No association was detected between regular physical activity and CSF, as well as serum biomarkers.
Conclusion:
Regular physical activity is associated with favorable PD biomarker progression, indicating a potential disease-modifying effect.
Background
Irisin is a myokine that increases with leisure time physical activity (LTPA) and for which a cardiovascular protective role has been postulated. Our aim was to assess this role in the general population.
Methods
A cross-sectional analysis was performed in a large randomly selected population sample (n=2298 women and 1529 men). Apart from age and sex, we record anthropometrics (blood pressure, heart rate, obesity), lifestyle (LTPA, smoking, alcohol), and biochemical measurements (irisin, lipid profile, insulin resistance). Correlations and regression multivariate models were used to analyze the association of irisin levels with the studied factors.
Results
The variables more strongly and directly associated with irisin, adjusting the studied factors separately in women and men, were HOMA-2 (p=0.043 and p=0.001, respectively) and LTPA (p<0.001 and p=0.001, respectively). Also heart rate inversely (p=0.005 and p=0.002, respectively) and DBP directly (p<0.005 and p=0.045, respectively) were associated to irisin in both sexes. The waist/height ratio (p<0.001) was inversely associated to irisin only in women, and the alcohol drinking was directly associated (p=0.029) only in men.
Conclusion
We provide new findings for irisin, such as its association with DBP and with heart rate; furthermore, in women irisin is associated to abdominal obesity, and in men is associated to the alcohol intake. We also corroborate the association of irisin with LTPA and insulin resistance. The associations detected point towards a protective role of irisin in the maintenance of cardiometabolic health.
Background: This study was designed to investigate the relationship of irisin with the severity of Parkinson’s disease (PD) and dopamine (DOPA) uptake in patients with PD and to understand the role of irisin in PD and its potential value as a drug therapy.
Methods: The plasma levels of irisin and α-syn were measured by ELISA. Motor and nonmotor symptoms were assessed with related scales. DOPA uptake was measured with DOPA PET/MRI.
Results: The plasma level of α-syn and irisin in patients with PD increased and decreased gradually with the progression of the disease, respectively. There was a negative correlation between plasma α-syn and irisin levels in patients with PD. The level of irisin in plasma was negatively correlated with UPDRS-III scores and positively correlated with MOCA scores. The SORs of the ipsilateral and contralateral caudate nucleus, anterior putamen and posterior putamen in the high-Irisin group was significantly higher than those in the low-Irisin group, and irisin levels in the caudate nucleus, anterior putamen, and posterior putamen contralateral to the affected limb were lower than those on the ipsilateral side. The level of irisin was positively correlated with the SORs of the ipsilateral and contralateral caudate nucleus and putamen in PD patients.
Conclusions: Irisin plays a neuroprotective role by decreasing the level of α-syn. Irisin is negatively correlated with the severity of motor symptoms and cognitive impairment. More importantly, irisin can improve DOPA uptake in the striatum of patients with PD, especially on the side contralateral to the affected limb.
Exercise benefits the human body in many ways. Irisin is secreted by muscle, increased with exercise, and conveys physiological benefits, including improved cognition and resistance to neurodegeneration. Irisin acts via αV integrins; however, a mechanistic understanding of how small polypeptides like irisin can signal through integrins is poorly understood. Using mass spectrometry and cryo-EM, we demonstrate that the extracellular heat shock protein 90α (eHsp90α) is secreted by muscle with exercise and activates integrin αVβ5. This allows for high-affinity irisin binding and signaling through an Hsp90α/αV/β5 complex. By including hydrogen/deuterium exchange data, we generate and experimentally validate a 2.98 Å RMSD irisin/αVβ5 complex docking model. Irisin binds very tightly to an alternative interface on αVβ5 distinct from that used by known ligands. These data elucidate a non-canonical mechanism by which a small polypeptide hormone like irisin can function through an integrin receptor.
Objective:
Cleavage of fibronectin type III domain-containing protein 5 (FNDC5), a membrane-bound precursor protein, would cleave into a myokine, irisin, which is also expressed in the liver. FNDC5/Irisin has been reported to play a critical role in maintaining glucose and lipid homeostasis in the liver and in combating liver fibrosis. Recently, several studies have shown that extracellular vesicles (EVs) derived from hepatic stellate cells (HSCs) could modulate liver fibrosis; however, there is a large gap in understanding whether inhibition of fibrogenic EVs derived from HSCs could alleviate the progression of liver fibrosis. Here, we investigated the role of FNDC5/irisin in liver fibrosis and the mechanism of its inhibitory role in the release of HSC-derived fibrogenic EVs.
Methods:
Experiments were performed in wild-type and FNDC5-/- mice, primary mouse HSCs, and human hepatic stellate cell line (LX2). Mice were treated with carbon tetrachloride (CCl4) or bile duct ligation (BDL) to induce liver fibrosis. EVs derived from HSCs were purified and injected intraperitoneally into mice.
Results:
Our results showed that FNDC5 deficiency exacerbated CCl4-induced liver fibrosis and activation of HSCs in mice. Moreover, fibrogenic EVs derived from PDGF-BB-treated HSCs promoted HSC migration in vitro and liver fibrosis in vivo. However, administration of irisin, a cleavage of FNDC5, inhibited the release of fibrogenic EVs and activation of HSCs by promoting ubiquitylation degradation of Rab27b. In vivo, the promoting role of HSC-derived fibrogenic EVs in liver fibrosis was also reversed by irisin.
Conclusion:
All these results demonstrate that FNDC5/irisin is a novel therapeutic agent for chronic liver fibrosis.
Irisin, a myokine mainly secreted from contracted skeleton muscle, plays a profound role in bone formation and remodeling. Although irisin has been revealed to elevate bone mass in vivo, details whether there is a dose-dependent relationship between irisin and bone formation remain unclear. In this study, we explored the dose-dependent effects of irisin on osteoblast proliferation and differentiation. Our results first demonstrated a remarkable increase in cell proliferation rate and viability in response to elevated concentrations of r-irisin, which was further enhanced over time. Notably, this increase was subject to complex dose-response relationships as the proliferation-enhancing effects of r-irisin may have a saturation point between 10 ng/ml and 100 ng/ml. Furthermore, we determined that 1, 10, and 100 ng/ml r-irisin were able to upregulate the expression of osteogenic transcription factors (Runx2, Osx, and Atf4), as well as osteogenic markers (Alp, Col1a1 and Spp1), albeit without significant difference among these 3 concentrations. Interestingly, nutrient-depleted osteoblasts and those with standard culture showed distinct responses to higher doses of irisin regarding osteogenic differentiation. Further investigation is required to uncover the molecular mechanisms underlying the observed tandem effects of irisin on osteogenesis.
Researchers have always concluded that results that do not support the hypothesis as unimportant, unworthy, or simply not good enough for publication. However, negative findings are essential for the progress of science and its self-correcting nature. We also believe in the importance and indispensability of negative results. Therefore, in this review, we discussed the factors contributing to the publication bias of negative results and the problems to assess the factuality and validity of negative results. Moreover, we emphasized the importance of reporting negative results in cardiovascular research, including treatments, and suggest that the negative results could clarify previously controversial topics in the treatment of cardiovascular diseases and prompt the translation of research on precision cardiovascular disease prevention and treatment.
Air pollution consisting of fine particulate matter (PM2.5) can induce or aggravate pulmonary inflammatory injury. Irisin has been shown to inhibit inflammation and help to protect against acute kidney, lung or brain injury. However, the role of irisin in lung inflammation after exposure to PM2.5 remains unclear. The aim of this study was to investigate the effect and molecular mechanism of irisin supplementation on in vitro and in vivo models of PM2.5-induced acute lung injury（ALI). C57BL/6 mice and alveolar macrophage cell line (MH-S) were treated with PM2.5. Histopathological examination and FNDC5/ irisin immunofluorescence staining was performed on lung tissue sections. MH-S cell viability was determined by CCK-8 assay. The levels of Nod2, NF-κB p65 and NLRP3 were detected by qRT-PCR and western blotting. The levels of cytokines (IL-1β, IL-18 and TNF-α) were detected by ELISA. PM2.5 exposure induced increased secretion of pro-inflammatory factors and activation of Nod2, NF-κB p65 and NLRP3 as well as endogenous levels of irisin. In vivo and in vitro inflammation was alleviated by irisin supplementation. Irisin significantly decreased IL-1β, IL-18, and TNF-α production at both mRNA and protein level. Expression levels of Nod2, NF-κB p65, and NLRP3 were all significantly affected by irisin. In vivo the degree of pulmonary injury and inflammatory infiltration was weakened after irisin administration. In vitro, irisin could inhibit the activation of the NLRP3 inflammasome for a sustained period of 24 h, and its inhibitory ability was gradually enhanced. In conclusion, our findings indicate that irisin can modulate the inflammatory injury of lung tissue caused by PM2.5 through the Nod2/NF-κB signaling pathway, suggesting that irisin can be a candidate for the therapeutic or preventive intervention in acute lung inflammation.
How exercise elicits systemic metabolic benefits in both muscles and non-contractile tissues is unclear. Autophagy is a stress-induced lysosomal degradation pathway that mediates protein and organelle turnover and metabolic adaptation. Exercise activates autophagy in not only contracting muscles but also non-contractile tissues including the liver. However, the role and mechanism of exercise-activated autophagy in non-contractile tissues remain mysterious. Here, we show that hepatic autophagy activation is essential for exercise-induced metabolic benefits. Plasma or serum from exercised mice is sufficient to activate autophagy in cells. By proteomic studies, we identify fibronectin (FN1), which was previously considered as an extracellular matrix protein, as an exercise-induced, muscle-secreted, autophagy-inducing circulating factor. Muscle-secreted FN1 mediates exercise-induced hepatic autophagy and systemic insulin sensitization via the hepatic receptor α5β1 integrin and the downstream IKKα/β-JNK1-BECN1 pathway. Thus, we demonstrate that hepatic autophagy activation drives exercise-induced metabolic benefits against diabetes via muscle-secreted soluble FN1 and hepatic α5β1 integrin signaling.
Irisin is a 112 amino acid peptide hormone cleaved from the fibronectin type III domain-containing protein. Irisin is highly conserved across vertebrates, suggesting evolutionarily conserved common functions among domestic animals. These functions include the browning of white adipose tissue and increased energy expenditure. Irisin has been detected and studied primarily in plasma, serum, and skeletal muscle, but has also been found in adipose tissue, liver, kidney, lungs, cerebrospinal fluid, breast milk, and saliva. This wider tissue presence of irisin suggests additional functions beyond its role as a myokine in regulating energy use. We are beginning to understand irisin in domestic animals. The goal of this review is to provide an up-to-date commentary on irisin structure, tissue distribution, and functions across vertebrates, especially mammals of importance in veterinary medicine. Irisin could be explored as a potential candidate for developing therapeutic agents and biomarkers in domestic animal endocrinology.
In a previous report it was shown that mammalian ribosomes were capable of initiating translation at a non-AUG triplet when the initiation codon of mouse dihydrofolate reductase (dhfr) was mutated to ACG (Peabody, D.S. (1987) J. Biol. Chem. 262, 11847–11851). In order to assess the capacity of the mammalian translation apparatus to initiate at other non-AUG triplets, the initiator AUG of dihydrofolate reductase was converted to GUG, UUG, CUG, AGG, AAG, AUA, AUC, and AUU. These represent (with ACG) all the possible triplets that differ from AUG by only one nucleotide. The ability of each mutant to produce dihydrofolate reductase was assessed by in vitro transcription/translation of the mutant dhfr sequences under control of the bacteriophage SP6 promoter. Each of the triplets (with the exceptions of AGG and AAG) was able to direct the synthesis of apparently normal dihydrofolate reductase. Incorporation of [³⁵S]tRNAifMet into the products of in vitro translation indicates that in each case the non-AUG triplet is able to direct initiation of the polypeptide chain with methionine. The mutant dhfr sequences were also inserted into the mammalian expression vector SVGT5 for expression in cultured monkey cells. The hierarchy of relative translation efficiencies was similar in vivo and in vitro.
The myokine irisin is supposed to be cleaved from a transmembrane precursor, FNDC5 (fibronectin type III domain containing 5), and to mediate beneficial effects of exercise on human metabolism. However, evidence for irisin circulating in blood is largely based on commercial ELISA kits which are based on polyclonal antibodies (pAbs) not previously tested for cross-reacting serum proteins. We have analyzed four commercial pAbs by Western blotting, which revealed prominent cross-reactivity with non-specific proteins in human and animal sera. Using recombinant glycosylated and non-glycosylated irisin as positive controls, we found no immune-reactive bands of the expected size in any biological samples. A FNDC5 signature was identified at ~20 kDa by mass spectrometry in human serum but was not detected by the commercial pAbs tested. Our results call into question all previous data obtained with commercial ELISA kits for irisin, and provide evidence against a physiological role for irisin in humans and other species.
Context:
Irisin has been proposed to be a myokine mediating the effect of exercise on adipocyte browning. The physiology of irisin in humans is not completely understood.
Objective:
To study the physiology of irisin in healthy individuals with different age and fitness levels and to explore the direct effects of irisin on muscle metabolism.
Design, setting, and subjects:
Treadmill exercise studies were conducted to measure circulating irisin at baseline and in response to exercise among old and young, physically active and sedentary individuals. Also, high- and moderate-intensity swimming was performed in adolescent men and women to study the effect of exercise intensity and the time course of irisin induction by acute bouts of exercise. Human myotubes were treated with recombinant irisin, and the effect on gene expression, cell signaling, and metabolism was examined.
Results:
Baseline circulating irisin was lower in old (vs young) and physically active (vs sedentary) subjects. Despite differences in basal levels, the percentage increase of irisin by acute bouts of exercise was not related to age or fitness level. The time course study revealed that circulating irisin increased immediately after high-intensity interval exercise and declined 1 hour thereafter. In vitro experiments showed that irisin facilitates glucose and lipid metabolism in human muscle through AMP kinase phosphorylation.
Conclusions:
Despite the differences in basal irisin levels, exercise-induced irisin secretion is independent of age or fitness level. Increased irisin can directly modulate muscle metabolism through AMP kinase activation.
Background:
Irisin, a recently discovered myokine, has been shown to induce browning of white adipose tissue, enhancing energy expenditure and mediating some of the beneficial effects of exercise. We aimed to estimate the time frame of changes in irisin levels after acute exercise and the effect of different exercise workloads and intensities on circulating irisin levels immediately post-exercise.
Methods:
In a pilot study, four healthy subjects (22.5±1.7 years) underwent maximal workload exercise (maximal oxygen consumption, VO2 max) and blood was drawn at prespecified intervals to define the time frame of pre- and post-exercise irisin changes over a 24-h period. In the main study, 35 healthy, non-smoking (23.0±3.3 years) men and women (n=20/15) underwent three exercise protocols ≥48-h apart, in random order: i) maximal workload (VO2 max); ii) relative workload (70% of VO2 max/10 min); and iii) absolute workload (75 W/10 min). Blood was drawn immediately pre-exercise and 3 min post-exercise.
Results:
In the pilot study, irisin levels increased by 35% 3 min post-exercise, then dropped and remained relatively constant. In the main study, irisin levels post-exercise were significantly higher than those of pre-exercise after all workloads (all, P<0.001). Post-to-pre-exercise differences in irisin levels were significantly different between workloads (P=0.001), with the greatest increase by 34% following maximal workload (P=0.004 vs relative and absolute).
Conclusions:
Circulating irisin levels were acutely elevated in response to exercise, with a greater increase after maximal workload. These findings suggest that irisin release could be a function of muscle energy demand. Future studies need to determine the underlying mechanisms of irisin release and explore irisin's therapeutic potential.
Key points
Considerable controversy exists regarding the role of irisin, a putative exercise‐induced myokine, in human metabolism.
We therefore studied irisin and its precursor Fndc5 in obesity, type 2 diabetes and exercise.
Complex clinical studies combined with cell culture work revealed that Fndc5 /irisin was decreased in type 2 diabetes in vivo , but not in muscle cells in vitro , indicating that diabetes‐related factor(s) regulate Fndc5 /irisin in vivo .
Several attributes of type 2 diabetes, such as hyperglycaemia, triglyceridaemia, visceral adiposity and extramyocellular lipid deposition were negatively associated with adipose tissue Fndc5 mRNA and circulating irisin. Moreover, mimicking diabetic status in vitro by treating muscle cells with palmitate and glucose lowered Fndc5 mRNA.
Neither exercise training nor an acute exercise bout modulated circulating irisin or muscle Fndc5 expression. However, the associations between intensity of habitual physical activity, muscle volume, strength, contractility and circulating irisin provide a link between irisin and positive outcomes of increased physical activity.
Abstract
Irisin was identified as a myokine secreted by contracting skeletal muscle, possibly mediating some exercise health benefits via ‘browning’ of white adipose tissue. However, a controversy exists concerning irisin origin, regulation and function in humans. Thus, we have explored Fndc5 gene and irisin protein in two clinical studies: (i) a cross‐sectional study (effects of type 2 diabetes (T2D) in drug‐naive men) and (ii) an intervention study (exercise effects in sedentary, overweight/obese individuals). Glucose tolerance and insulin sensitivity were assessed. Maximal aerobic capacity and muscle strength were measured before and after training. Body composition (magnetic resonance imaging), muscle and liver fat content ( ¹ H‐magnetic resonance spectroscopy (MRS)) and in vivo muscle metabolism ( ³² P‐MRS) were determined. Skeletal muscle and subcutaneous abdominal adipose tissue samples were taken in the fasted state and during euglycaemic hyperinsulinaemia (adipose tissue) and before/after exercise training (muscle). We found that muscle Fndc5 mRNA was increased in prediabetes but not T2D. Fndc5 in adipose tissue and irisin in plasma were reduced in T2D by 40% and 50%, respectively. In contrast, T2D‐derived myotubes expressed/secreted the highest levels of Fndc5 /irisin. Neither hyperinsulinaemia (adipose tissue/plasma) nor exercise (muscle/plasma) affected Fndc5 /irisin in vivo . Circulating irisin was positively associated with muscle mass, strength and metabolism and negatively with fasting glycaemia. Glucose and palmitate decreased Fndc5 mRNA in myotubes in vitro . We conclude that distinct patterns of Fndc5 /irisin in muscle, adipose tissue and circulation, and concordant in vivo down‐regulation in T2D, indicate that irisin might distinguish metabolic health and disease. Moreover, Fndc5 /irisin was discordantly regulated in diabetic muscle and myotubes in vitro , suggesting that whole body factors, such as glucose and fatty acids, might be important for irisin regulation. Exercise did not affect Fndc5 /irisin. However, irisin was positively linked to muscle mass, strength and metabolism, pointing to common regulatory factors and/or the potential for irisin to modify muscle phenotype.
Exercise can improve cognitive function and has been linked to the increased expression of brain-derived neurotrophic factor (BDNF). However, the underlying molecular mechanisms driving the elevation of this neurotrophin remain unknown. Here we show that FNDC5, a previously identified muscle protein that is induced in exercise and is cleaved and secreted as irisin, is also elevated by endurance exercise in the hippocampus of mice. Neuronal Fndc5 gene expression is regulated by PGC-1α, and Pgc1a(-/-) mice show reduced Fndc5 expression in the brain. Forced expression of FNDC5 in primary cortical neurons increases Bdnf expression, whereas RNAi-mediated knockdown of FNDC5 reduces Bdnf. Importantly, peripheral delivery of FNDC5 to the liver via adenoviral vectors, resulting in elevated blood irisin, induces expression of Bdnf and other neuroprotective genes in the hippocampus. Taken together, our findings link endurance exercise and the important metabolic mediators, PGC-1α and FNDC5, with BDNF expression in the brain.
Irisin, a newly discovered, PGC-1α dependent myokine, has recently been shown to increase in circulation in response to sprint exercise. This study examined the effect of prolonged exercise on irisin concentrations in young men (n=7) as well as in young women (n=5) during different stages of the menstrual cycle. Seven young men completed 90 min of treadmill exercise at 60% of VO2max and a resting control trial. Five women completed the same exercise protocol in two different trials: during the early follicular phase and mid-luteal phase of the menstrual cycle. Blood samples were collected and analyzed for irisin concentrations immediately before exercise, at 54 and 90 min of exercise, and at 20 min of recovery (R20). Findings revealed that by 54 min of a 90 min treadmill exercise protocol at 60% of VO2max, irisin concentrations significantly increased 20.4% in young men and 20.3% as well as 24.6% in young women during the early follicular and mid-luteal phases of the menstrual cycle, respectively. However, by 90 min of exercise as well as R20, irisin concentrations were no longer elevated. Stage of the menstrual cycle did not affect responses in young women. Findings indicate that prolonged aerobic exercise produces a transient increase in irisin concentrations during the first hour of exercise for both genders and suggest that this form of moderate exercise may be helpful in improving fat metabolism.
FNDC5 (fibronectin domain-containing [protein] 5) was initially discovered and characterized by two groups in 2002. In 2011 FNDC5 burst into prominence as the parent of irisin, a small protein containing the fibronectin type III domain. Irisin was proposed to be secreted by skeletal muscle cells in response to exercise, and to circulate to fat tissue where it induced a transition to brown fat. Since brown fat results in dissipation of energy, this pathway is of considerable interest for metabolism and obesity. Here I review the original discoveries of FNDC5 and the more recent discovery of irisin. I note in particular three problems in the characterization of irisin: the antibodies used to detect irisin in plasma lack validity; the recombinant protein used to demonstrate activity in cell culture was severely truncated; and the degree of shedding of soluble irisin from the cell surface has not been quantitated. The original discovery proposing that FNDC5 may be a transmembrane receptor may deserve a new look.
Brown adipose tissue has gained interest as a potential target to treat obesity and metabolic diseases. Irisin is a newly identified hormone secreted from skeletal muscle enhancing browning of white fat cells, which improves systemic metabolism by increasing energy expenditure in mice. The discovery of irisin raised expectations of its therapeutic potential to treat metabolic diseases. However, the effect of irisin in humans is unclear. Analyses of genomic DNA, mRNA and expressed sequence tags revealed that FNDC5, the gene encoding the precursor of irisin, is present in rodents and most primates, but shows in humans a mutation in the conserved start codon ATG to ATA. HEK293 cells transfected with a human FNDC5 construct with ATA as start codon resulted in only 1% full-length protein compared to human FNDC5 with ATG. Additionally, in vitro contraction of primary human myotubes by electrical pulse stimulation induced a significant increase in PGC1α mRNA expression. However, FNDC5 mRNA level was not altered. FNDC5 mRNA expression in muscle biopsies from two different human exercise studies was not changed by endurance or strength training. Preadipocytes isolated from human subcutaneous adipose tissue exhibited differentiation to brite human adipocytes when incubated with bone morphogenetic protein (BMP) 7, but neither recombinant FNDC5 nor irisin were effective. In conclusion, our findings suggest that it is rather unlikely that the beneficial effect of irisin observed in mice can be translated to humans.
Irisin, a hormone secreted by myocytes induced in exercise, acts as a muscle-derived energy-expenditure signal that binds to undetermined receptors on the white adipose tissue surface, stimulating its browning and uncoupling protein 1 (UCP1) expression. The purpose of this study was to assess the effect of an intradialytic resistance exercise training program (RETP) on plasma irisin levels of hemodialysis (HD) patients and compare the baseline plasma irisin levels of HD patients to healthy subjects. This longitudinal study enrolled 26 patients undergoing HD (50% men, 44.8±14.1 years, body mass index (BMI) 23.5±3.9 kg/m2). The healthy subjects group consisted of 11 women and 7 men with mean age of 50.9±6.6 years and BMI, 24.2±2.7 kg/m2. Anthropometric and biochemistry parameters (Irisin by Enzyme-Linked Immunosorbent Assay) were measured at the baseline and after 6 months of RETP (in both lower limbs). There was no difference regarding gender, age, and BMI between HD patients and healthy subjects. Plasma irisin levels in HD patients were lower than in healthy subjects (71.0±41.6 vs. 101.3±12.5 ng/ml, p<0.05). Although the muscle mass increased in consequence of exercise [evaluated by arm muscle area from 27.9 (24.1) to 33.1 (19.0) cm2], plasma irisin did not differ significantly after exercises (71.0±41.6 vs. 73.3±36.0 ng/ml). HD patients seem to have lower plasma irisin when compared to healthy subjects. Moreover, a resistance exercise training program was unable to augment plasma irisin despite increasing muscle mass.
Exercise benefits a variety of organ systems in mammals, and some of the best-recognized effects of exercise on muscle are mediated by the transcriptional co-activator PPAR-γ co-activator-1 α (PGC1-α). Here we show in mouse that PGC1-α expression in muscle stimulates an increase in expression of FNDC5, a membrane protein that is cleaved and secreted as a newly identified hormone, irisin. Irisin acts on white adipose cells in culture and in vivo to stimulate UCP1 expression and a broad program of brown-fat-like development. Irisin is induced with exercise in mice and humans, and mildly increased irisin levels in the blood cause an increase in energy expenditure in mice with no changes in movement or food intake. This results in improvements in obesity and glucose homeostasis. Irisin could be therapeutic for human metabolic disease and other disorders that are improved with exercise.
The UniProt Knowledgebase (UniProtKB) acts as a central hub of protein knowledge by providing a unified view of protein sequence and functional information. Manual and automatic annotation procedures are used to add data directly to the database while extensive cross-referencing to more than 120 external databases provides access to additional relevant information in more specialized data collections. UniProtKB also integrates a range of data from other resources. All information is attributed to its original source, allowing users to trace the provenance of all data. The UniProt Consortium is committed to using and promoting common data exchange formats and technologies, and UniProtKB data is made freely available in a range of formats to facilitate integration with other databases.
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In eukaryotes, it is generally assumed that translation initiation occurs at the AUG codon closest to the messenger RNA 5′
cap. However, in certain cases, initiation can occur at codons differing from AUG by a single nucleotide, especially the codons
CUG, UUG, GUG, ACG, AUA and AUU. While non-AUG initiation has been experimentally verified for a handful of human genes, the
full extent to which this phenomenon is utilized—both for increased coding capacity and potentially also for novel regulatory
mechanisms—remains unclear. To address this issue, and hence to improve the quality of existing coding sequence annotations,
we developed a methodology based on phylogenetic analysis of predicted 5′ untranslated regions from orthologous genes. We
use evolutionary signatures of protein-coding sequences as an indicator of translation initiation upstream of annotated coding
sequences. Our search identified novel conserved potential non-AUG-initiated N-terminal extensions in 42 human genes including
VANGL2, FGFR1, KCNN4, TRPV6, HDGF, CITED2, EIF4G3 and NTF3, and also affirmed the conservation of known non-AUG-initiated
extensions in 17 other genes. In several instances, we have been able to obtain independent experimental evidence of the expression
of non-AUG-initiated products from the previously published literature and ribosome profiling data.
Skyline is a Windows client application for targeted proteomics method creation and quantitative data analysis. It is open source and freely available for academic and commercial use. The Skyline user interface simplifies the development of mass spectrometer methods and the analysis of data from targeted proteomics experiments performed using selected reaction monitoring (SRM). Skyline supports using and creating MS/MS spectral libraries from a wide variety of sources to choose SRM filters and verify results based on previously observed ion trap data. Skyline exports transition lists to and imports the native output files from Agilent, Applied Biosystems, Thermo Fisher Scientific and Waters triple quadrupole instruments, seamlessly connecting mass spectrometer output back to the experimental design document. The fast and compact Skyline file format is easily shared, even for experiments requiring many sample injections. A rich array of graphs displays results and provides powerful tools for inspecting data integrity as data are acquired, helping instrument operators to identify problems early. The Skyline dynamic report designer exports tabular data from the Skyline document model for in-depth analysis with common statistical tools. Availability: Single-click, self-updating web installation is available at http://proteome.gs.washington.edu/software/skyline. This website also provides access to instructional videos, a support board, an issues list and a link to the source code project. Contact: [email protected]
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The initiator AUC of the mouse dihydrofolate reductase gene (dhfr) was converted to ACG by site-directed mutagenesis and assayed for expression in cultured monkey cells using an SV40 recombinant called SVGT5dhfr26m2. Synthesis of apparently full-length dihydrofolate reductase (DHFR) protein was significantly reduced compared to wild-type, but not entirely abolished, suggesting that the ACG triplet was being utilized for translation initiation. In addition, a truncated form of DHFR was produced, apparently by initiation at the next in-frame AUG downstream. This result was confirmed in vitro. Transcripts of the dhfr sequence were produced by SP6 RNA polymerase in the presence of m7GpppG and translated in vitro using reticulocyte lysates and wheat germ extracts. The results paralleled those observed in vivo. Synthesis of full-length DHFR was reduced, but not eliminated, and a new species was produced by initiation at an internal site. Amino acid sequence analysis of the products of in vitro translation demonstrated that translation does indeed initiate at the ACG triplet and that it initiates with methionine. Additional mutations were introduced which altered the sequence context of the ACG triplet. Mutation of the translation initiation consensus sequence by substitution of the A residue at position -3, or of the G at +4 resulted in a significant decrease in initiation at the ACG and an increase in the level of the internal initiation product. Thus, translation initiation at a non-AUG triplet depends on a favorable sequence context.
A need exists for technologies that permit the direct quantification of differences in protein and posttranslationally modified protein expression levels. Here we present a strategy for the absolute quantification (termed AQUA) of proteins and their modification states. Peptides are synthesized with incorporated stable isotopes as ideal internal standards to mimic native peptides formed by proteolysis. These synthetic peptides can also be prepared with covalent modifications (e.g., phosphorylation, methylation, acetylation, etc.) that are chemically identical to naturally occurring posttranslational modifications. Such AQUA internal standard peptides are then used to precisely and quantitatively measure the absolute levels of proteins and posttranslationally modified proteins after proteolysis by using a selected reaction monitoring analysis in a tandem mass spectrometer. In the present work, the AQUA strategy was used to (i) quantify low abundance yeast proteins involved in gene silencing, (ii) quantitatively determine the cell cycle-dependent phosphorylation of Ser-1126 of human separase protein, and (iii) identify kinases capable of phosphorylating Ser-1501 of separase in an in vitro kinase assay. The methods described here represent focused, alternative approaches for studying the dynamically changing proteome.
Although previous studies have already shown that both cytoplasmic and mitochondrial activities of glycyl-tRNA synthetase
are provided by a single gene, GRS1,in the yeast Saccharomyces cerevisiae, the mechanism by which this occurs remains unclear. Evidence presented here indicates that this bifunctional property is
actually a result of two distinct translational products alternatively generated from a single transcript of this gene. Except
for an amino-terminal 23-amino acid extension, these two isoforms have the same polypeptide sequence and function exclusively
in their respective compartments under normal conditions. Reporter gene assays further suggest that this leader peptide can
function independently as a mitochondrial targeting signal and plays the major role in the subcellular localization of the
isoforms. Additionally, whereas the short protein is translationally initiated from a traditional AUG triplet, the longer
isoform is generated from an upstream inframe UUG codon. To our knowledge, GRS1 appears to be the first example in the yeast wherein a functional protein isoform is initiated from a naturally occurring
non-AUG codon. The results suggest that non-AUG initiation might be a mechanism existing throughout all kingdoms.
Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.
Mass spectrometry (MS)-based proteomics measures peptides derived from proteins by proteolytic cleavage. Before performing the analysis by matrix-assisted laser desorption/ionization-tandem mass spectrometry (MALDI-MS/MS), nanoelectrospray-MS/MS (NanoES-MS/MS) or liquid chromatography-MS/MS (LC-MS/MS), the peptide mixtures need to be cleaned, concentrated and often selectively enriched or pre-fractionated, for which we employ simple, self-made and extremely economical stop-and-go-extraction tips (StageTips). StageTips are ordinary pipette tips containing very small disks made of beads with reversed phase, cation-exchange or anion-exchange surfaces embedded in a Teflon mesh. The fixed nature of the beads allows flexible combination of disks with different surfaces to obtain multi-functional tips. Disks containing different surface functionalities and loose beads such as titania and zirconia for phosphopeptide enrichment can be combined. Incorporation into an automated workflow has also been demonstrated. Desalting and concentration takes approximately 5 min while fractionation or enrichment takes approximately 30 min.
The processed pseudogenes reported to date fall into three categories: those that are a complete copy of the mRNA transcribed from the functional gene, those that are only a partial copy of the corresponding mRNA, and those that contain sequences in addition to those expected to be present in the mRNA. The general structural characteristics of these processed pseudogenes include the complete lack of intervening sequences found in the functional counterparts, a poly A tract at the 3' end, and direct repeats flanking the pseudogene sequence. In all the cases studied, these pseudogenes have been found to be on a different chromosome from their functional counterpart. These characteristics have led investigators to suggest that an RNA intermediate, in many cases the mRNA of the functional gene, is involved in the production of these pseudogenes. The mechanism by which processed pseudogenes arose involves the integration of the mRNA, or its cDNA copy, into a staggered chromosome break, followed by DNA synthesis and repair. I suggest that all the transcripts that gave rise to these pseudogenes were actually produced in the germ line cell. The transcripts that gave rise to the processed pseudogenes that are direct copies of the corresponding mRNA resulted from RNA polymerase II transcription of the functional counterpart. Pseudogenes that are not a direct copy of the corresponding mRNA may have resulted from RNA polymerase III transcription. If this is indeed the case, one need not postulate the involvement of retroviruses to explain the presence of processed pseudogenes corresponding to genes that are not expressed in the germ line. Following the integration event, processed pseudogenes can no longer be transcribed to produce the functional mRNA from which they arose. This inability to be transcribed by RNA polymerase II is not surprising considering that processed pseudogenes seem to be randomly integrated into the genome. Therefore, integration of a processed pseudogene such that RNA polymerase II transcriptional promoters are correctly positioned 5' to the resultant pseudogene is an unlikely event. The presence of processed pseudogenes seems peculiar to mammals. In fact, evolutionary studies indicate that processed pseudogenes are of relatively recent origin. In fact, at least one processed pseudogene, the human DHFR psi 1, has been formed so recently that it is polymorphic.
Aim:
Irisin is first discovered as a potential mediator of obesity related energy homeostasis. Recent studies indicate that irisin is associated with endothelial dysfunction and atherosclerosis in patients with type 2 diabetes. Our objective was to examine the relationship between irisin and urinary albumin excretion in patients with type 2 diabetes.
Methods:
100 newly diagnosed patients with type 2 diabetes and 100 healthy subjects were selected. Serum irisin levels were measured by ELISA, and urine albumin was measured by radioimmunoassay. High resolution ultrasound was used to measure brachial artery diameter at rest, after reactive hyperemia (flow-mediated arterial dilation, FMD) and after sublingual glyceryltrinitrate.
Results:
Patients with type 2 diabetes presented decreased irisin levels when compared to controls (14.12±3.93 versus 28.98±2.56ng/ml, P=0.015).Serum irisin levels in the microalbuminuric and macroalbuminuria subgroup were 9.89±1.56ng/ml and 5.67±1.89ng/ml, respectively, which were significantly lower than those in the normoalbuminuria (15.97±3.12ng/ml). In comparison to microalbuminuric subgroup, macroalbuminuria subgroup had lower levels of irisin. By dividing the distribution of serum irisin levels into quartiles, FMD was increased gradually with the increase of serum irisin levels (P<0.001). Multiple stepwise linear regression analysis showed that FMD (β=0.75, P=0.002), 2-hBG (β=-0.25, P=0.038) and UAE (β=-0.87, P=0.008) were significantly associated with irisin. Pearson's correlation analyses showed a negative correlation between irisin and logUAE (r=-0.57) and between FMD and logUAE (r=-0.47), and positive correlations between irisin and FMD (r=0.51).
Conclusions:
Decreased plasma levels of irisin seem to be associated with UAE and FMD in patients with type 2 diabetes.
Exercise represents a major challenge to whole-body homeostasis provoking widespread perturbations in numerous cells, tissues, and organs that are caused by or are a response to the increased metabolic activity of contracting skeletal muscles. To meet this challenge, multiple integrated and often redundant responses operate to blunt the homeostatic threats generated by exercise-induced increases in muscle energy and oxygen demand. The application of molecular techniques to exercise biology has provided greater understanding of the multiplicity and complexity of cellular networks involved in exercise responses, and recent discoveries offer perspectives on the mechanisms by which muscle "communicates" with other organs and mediates the beneficial effects of exercise on health and performance.
Aim:
Type 2 diabetes mellitus (T2DM) is not simply a disease of hyperglycemia, but also is an inflammatory disorder. This study aimed to observe the expression of inflammation-related factors in elderly T2DM patients with or without macrovascular disease (MVD).
Patients and methods:
A total of 64 T2DM patients participated in this study, including 31 patients with MVD (group A) and 33 patients without MVD (group B); and 30 healthy volunteers were recruited as normal control (group C). The levels of serum irisin, retinol-binding protein 4 (RBP4) and adiponectin expression were all detected and compared between groups.
Results:
The demographic and clinical characteristics were comparable between T2DM patients and healthy volunteers. For patients in group A, the serum levels of irisin, RBP4 and adiponectin were 12.05 ± 2.12 pg/mL, 2.13 ± 0.83 µg/mL and 45.65±20.13 ng/mL, respectively. While the corresponding parameters were 26.11 ± 4.09 pg/ml, 1.54 ± 0.54 µg/ml and 57.93 ± 23.47 ng/mL for patients in group B; and were 40.25 ± 2.73 pg/mL, 0.98 ± 0.36 µg/mL and 60.03 ± 20.26 ng/mL for healthy volunteers in group C, respectively. As compared to healthy volunteers, the levels of irisin, RBP4 and adiponectin were all significantly changed in T2DM patients; and the difference in irisin, RBP4 and adiponectin between T2DM patients with and without MVD were all significant (p = 0.000, p = 0.001, and p = 0.029, respectively). Multivariate regression analysis showed that irisin and RBP4 are both independent predictors for MVD in T2DM patients.
Conclusions:
Inflammatory disorder is significantly in T2DM patients with MVD, and serum irisin and RBP4 would be reasonable new markers of MVD.
Rediscovery of cold-activated brown adipose tissue (BAT) in humans has boosted research interest in identifying BAT activators for metabolic benefits. Of particular interest are cytokines capable of fat browning. Irisin, derived from FNDC5, is an exercise-induced myokine that drives brown-fat-like thermogenesis in murine white fat. Here we explored whether cold exposure is an afferent signal for irisin secretion in humans and compared it with FGF21, a brown adipokine in rodents. Cold exposure increased circulating irisin and FGF21. We found an induction of irisin secretion proportional to shivering intensity, in magnitude similar to exercise-stimulated secretion. FNDC5 and/or FGF21 treatment upregulated human adipocyte brown fat gene/protein expression and thermogenesis in a depot-specific manner. These results suggest exercise-induced irisin secretion could have evolved from shivering-related muscle contraction, serving to augment brown fat thermogenesis in concert with FGF21. Irisin-mediated muscle-adipose crosstalk may represent a thermogenic, cold-activated endocrine axis that is exploitable in obesity therapeutics development.
The recently described myokine, irisin is cleaved from fibronectin type III domain containing protein 5 (FNDC5) and has been proposed to be secreted upon exercise to promote the browning of beige fat cells in white adipose tissue that results in enhanced thermogenesis and increased energy expenditure. The initial studies suggested irisin as a treatment option for obesity and associated diseases such as type 2 diabetes mellitus and stimulated further research. However, the results of subsequent studies investigating the regulation of irisin by different types of exercise are partly conflicting and effects were only shown in highly selective patient populations so far. Moreover, other parameters like body weight or fat free mass were shown to influence irisin adding more complexity to the mechanisms regulating this hormone. The present review will describe the discovery of irisin, its potential role in adipose tissue-mediated thermogenesis, its regulation by exercise and lastly, discuss current controversies and highlight gaps of knowledge to be filled by future studies.
Irisin was first identified as a peroxisome proliferator-activated receptor γ co-activator-1 α (PGC-1α)-dependent myokine with the potential to induce murine brown-fat-like development of white adipose tissue. In humans, the regulatory effect of training on muscle FNDC5 mRNA expression and subsequently irisin levels in plasma is more controversial. We recruited 26 inactive men (13 normoglycaemic and normal weight = controls, and 13 slightly hyperglycaemic and overweight = prediabetes group) aged 40-65 y for a 12 w intervention of combined endurance- and strength-training with 4 sessions of training/week. Before and after the 12 w intervention period, participants were exposed to an acute endurance work-load of 45 min at 70% of VO2 max, and muscle biopsies were taken prior to and after exercise. Skeletal muscles mRNA for PGC1A and FNDC5 correlated and both PGC1A and FNDC5 mRNA levels increased after 12 w of training in both control and prediabetes subjects. Circulating irisin was reduced in response to 12 w of training, and was increased acutely (~1.2-fold) just after acute exercise. Plasma concentration of irisin was higher in prediabetes subjects compared to controls. There were little effects of 12 w of training on selected browning genes in subcutaneous adipose tissue. UCP1 mRNA did not correlate with FNDC5 expression in subcutaneous adipose tissue or skeletal muscle or with irisin levels in plasma. We observed no enhancing effect of long-term training on circulating irisin levels, and little or no effect of training on browning of subcutaneous white adipose tissue in humans. This article is protected by copyright. All rights reserved.
Significant progress has been made in understanding the neurobiological mechanisms through which exercise protects and restores the brain. In this feature review, we integrate animal and human research, examining physical activity effects across multiple levels of description (neurons up to inter-regional pathways). We evaluate the influence of exercise on hippocampal structure and function, addressing common themes such as spatial memory and pattern separation, brain structure and plasticity, neurotrophic factors, and vasculature. Areas of research focused more within species, such as hippocampal neurogenesis in rodents, also provide crucial insight into the protective role of physical activity. Overall, converging evidence suggests exercise benefits brain function and cognition across the mammalian lifespan, which may translate into reduced risk for Alzheimer's disease (AD) in humans.
Effective immune surveillance by cytotoxic T cells requires newly synthesized polypeptides for presentation by major histocompatibility
complex (MHC) class I molecules. These polypeptides are produced not only from conventional AUG-initiated, but also from cryptic
non–AUG-initiated, reading frames by distinct translational mechanisms. Biochemical analysis of ribosomal initiation complexes
at CUG versus AUG initiation codons revealed that cells use an elongator leucine-bound transfer RNA (Leu-tRNA) to initiate
translation at cryptic CUG start codons. CUG/Leu-tRNA initiation was independent of the canonical initiator tRNA (AUG/Met-tRNAiMet) pathway but required expression of eukaryotic initiation factor 2A. Thus, a tRNA-based translation initiation mechanism
allows non–AUG-initiated protein synthesis and supplies peptides for presentation by MHC class I molecules.
A method to correlate the uninterpreted tandem mass spectra of peptides produced under low energy (10–50 eV) collision conditions with amino acid sequences in the Genpept database has been developed. In this method the protein database is searched to identify linear amino acid sequences within a mass tolerance of ± 1 u of the precursor ion molecular weight. A cross-correlation function is then used to provide a measurement of similarity between the mass-to-charge ratios for the fragment ions predicted from amino acid sequences obtained from the database and the fragment ions observed in the tandem mass spectrum. In general, a difference greater than 0.1 between the normalized cross-correlation functions of the first- and second-ranked search results indicates a successful match between sequence and spectrum. Searches of species-specific protein databases with tandem mass spectra acquired from peptides obtained from the enzymatically digested total proteins of E. coli and S. cerevisiae cells allowed matching of the spectra to amino acid sequences within proteins of these organisms. The approach described in this manuscript provides a convenient method to interpret tandem mass spectra with known sequences in a protein database.
The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. Here we present a suite of techniques, based on ribosome profiling (the deep sequencing of ribosome-protected mRNA fragments), to provide genome-wide maps of protein synthesis as well as a pulse-chase strategy for determining rates of translation elongation. We exploit the propensity of harringtonine to cause ribosomes to accumulate at sites of translation initiation together with a machine learning algorithm to define protein products systematically. Analysis of translation in mouse embryonic stem cells reveals thousands of strong pause sites and unannotated translation products. These include amino-terminal extensions and truncations and upstream open reading frames with regulatory potential, initiated at both AUG and non-AUG codons, whose translation changes after differentiation. We also define a class of short, polycistronic ribosome-associated coding RNAs (sprcRNAs) that encode small proteins. Our studies reveal an unanticipated complexity to mammalian proteomes.
N-linked glycosylation is a biologically important protein modification, but only a small fraction of modification sites have been mapped. We developed a "filter aided sample preparation" (FASP)-based method in which glycopeptides are enriched by binding to lectins on the top of a filter and mapped 6367 N-glycosylation sites on 2352 proteins in four mouse tissues and blood plasma using high-accuracy mass spectrometry. We found 74% of known mouse N-glycosites and discovered an additional 5753 sites on a diverse range of proteins. Sites almost always have the N-!P-[S|T]-!P (where !P is not proline) and rarely the N-X-C motif or nonconsensus sequences. Combining the FASP approach with analysis of subcellular glycosite localization reveals that the sites always orient toward the extracellular space or toward the lumen of ER, Golgi, lysosome, or peroxisome. The N-glycoproteome contains a plethora of modification sites on factors important in development, organ-specific functions, and disease.
In a previous report it was shown that mammalian ribosomes were capable of initiating translation at a non-AUG triplet when the initiation codon of mouse dihydrofolate reductase (dhfr) was mutated to ACG (Peabody, D.S. (1987) J. Biol. Chem. 262, 11847-11851). In order to assess the capacity of the mammalian translation apparatus to initiate at other non-AUG triplets, the initiator AUG of dihydrofolate reductase was converted to GUG, UUG, CUG, AGG, AAG, AUA, AUC, and AUU. These represent (with ACG) all the possible triplets that differ from AUG by only one nucleotide. The ability of each mutant to produce dihydrofolate reductase was assessed by in vitro transcription/translation of the mutant dhfr sequences under control of the bacteriophage SP6 promoter. Each of the triplets (with the exceptions of AGG and AAG) was able to direct the synthesis of apparently normal dihydrofolate reductase. Incorporation of [35S]tRNAifMet into the products of in vitro translation indicates that in each case the non-AUG triplet is able to direct initiation of the polypeptide chain with methionine. The mutant dhfr sequences were also inserted into the mammalian expression vector SVGT5 for expression in cultured monkey cells. The hierarchy of relative translation efficiencies was similar in vivo and in vitro.
The processed pseudogenes reported to date fall into three categories: those that are a complete copy of the mRNA transcribed from the functional gene, those that are only a partial copy of the corresponding mRNA, and those that contain sequences in addition to those expected to be present in the mRNA. The general structural characteristics of these processed pseudogenes include the complete lack of intervening sequences found in the functional counterparts, a poly A tract at the 3' end, and direct repeats flanking the pseudogene sequence. In all the cases studied, these pseudogenes have been found to be on a different chromosome from their functional counterpart. These characteristics have led investigators to suggest that an RNA intermediate, in many cases the mRNA of the functional gene, is involved in the production of these pseudogenes. The mechanism by which processed pseudogenes arose involves the integration of the mRNA, or its cDNA copy, into a staggered chromosome break, followed by DNA synthesis and repair. I suggest that all the transcripts that gave rise to these pseudogenes were actually produced in the germ line cell. The transcripts that gave rise to the processed pseudogenes that are direct copies of the corresponding mRNA resulted from RNA polymerase II transcription of the functional counterpart. Pseudogenes that are not a direct copy of the corresponding mRNA may have resulted from RNA polymerase III transcription. If this is indeed the case, one need not postulate the involvement of retroviruses to explain the presence of processed pseudogenes corresponding to genes that are not expressed in the germ line. Following the integration event, processed pseudogenes can no longer be transcribed to produce the functional mRNA from which they arose. This inability to be transcribed by RNA polymerase II is not surprising considering that processed pseudogenes seem to be randomly integrated into the genome. Therefore, integration of a processed pseudogene such that RNA polymerase II transcriptional promoters are correctly positioned 5' to the resultant pseudogene is an unlikely event. The presence of processed pseudogenes seems peculiar to mammals. In fact, evolutionary studies indicate that processed pseudogenes are of relatively recent origin. In fact, at least one processed pseudogene, the human DHFR psi 1, has been formed so recently that it is polymorphic.
Proteins from silver-stained gels can be digested enzymatically and the resulting peptide analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nano-electrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.
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