Fibronectin Type III Domain Containing 5 Expression Research Articles

MYOKINE FUNDC5 COMPROMISES MITOCHONDRIAL DYSFUNCTION IN OSTEOARTHRITIS

Chondrocytic activity is downregulated by compromised autophagy and mitochondrial dysfunction to accelerate the development of osteoarthritis (OA). Irisin is a cleaved form of fibronectin type III domain containing 5 (FNDC5) and known to regulate bone turnover and muscle homeostasis. However, little is known about the role of irisin in chondrocytes and the development of OA. This talk will shed light on FNDC5 expression by human articular chondrocytes and compare normal and osteoarthritic cells with respect to autophagosome marker LC3-II and oxidative DNA damage marker 8-hydroxydeoxyguanosine (8-OHdG). In chondrocytes in vitro, irisin improves IL-1β-mediated growth inhibition, loss of specific cartilage markers and glycosaminoglycan production. Irisin further suppressed Sirt3 and UCP- 1 to improve mitochondrial membrane potential, ATP production, and catalase. This attenuated IL-1β-mediated production of reactive oxygen species, mitochondrial fusion, mitophagy, and autophagosome formation. In a surgical murine model of destabilization of the medial meniscus (DMM) intra-articular administration of irisin alleviates symptoms like cartilage erosion and synovitis. Furthermore, gait profiles of the treated limbs improved. In chondrocytes, irisin treatment upregulates autophagy, 8-OHdG and apoptosis in cartilage of DMM limbs. Loss of FNDC5 in chondrocytes correlates with human knee OA and irisin repressed inflammation-mediated oxidative stress and deficient extracellular matrix synthesis through retaining mitochondrial biogenesis and autophagy. The talk sheds new light on the chondroprotective actions of this myokine and highlights the remedial effects of irisin during progression of OA.

MiR-143-3p/FNDC5 axis: a novel regulator of insulin sensitivity.

Insulin resistance is a key hallmark in type 2 diabetes. In recent decades, there have been numerous studies of the causes of insulin resistance. microRNAs (miRNAs) participate in the regulation of multiple aspects of energy metabolism and miR-143-3p has been shown to induce insulin resistance. We aimed to predict the downstream targets of miR-143-3p and found a miR-143-3p binding site on the 3'-untranslated region of FNDC5 (Fibronectin type III domain containing 5) mRNA. We first confirmed that FNDC5 mRNA is a target of miR-143-3p using a double luciferase experiment, then constructed a prokaryotic expression system for the mature form of FNDC5, irisin, and expressed and purified irisin protein. We transfected a miR-143-3p mimic into HepG2-NTCP (Na+-taurocholate cotransporting polypeptide) cells using an NTCP targeting vector, then 24 h later, the glucose concentration of the culture medium, western blot analysis was analyzed. We next co-incubated the cells transfected with the miR-143-3p mimic with irisin for 12 h following by the assay of glucose uptake and AKT phosphorylation. The glucose concentration of the culture medium was higher than that associated with control miRNA-transfected cells (p < 0.01). Western blot analysis showed that the miR-143-3p mimic significantly reduced the expression of FNDC5 (p < 0.05) and the phosphorylation of AKT (Protein kinase B) (p < 0.05), implying impaired insulin signaling. which increased the glucose uptake (p < 0.0001) and AKT phosphorylation in the cells (p < 0.05). We conclude that FNDC5 is a direct target of miR-143-3p and that miR-143-3p induces insulin resistance by reducing its expression.

High-Intense Interval Training Prevents Cognitive Impairment and Increases the Expression of Muscle Genes FNDC5 and PPARGC1A in a Rat Model of Alzheimer's Disease.

Alzheimer's disease is the most common neurodegenerative disease in the world, characterized by the progressive loss of neuronal structure and function, whose main histopathological landmark is the accumulation of β-amyloid in the brain. It is well known that exercise is a neuroprotective factor and that muscles produce and release myokines that exert endocrine effects in inflammation and metabolic dysfunction. Thus, this work intends to establish the relationship between the benefits of exercise through the chronic training of HIIT on cognitive damage induced by the Alzheimer's model by the injection of β amyloid1-42. For this purpose, forty-eight male Wistar rats were divided into four groups: Sedentary Sham (SS), Trained Sham (ST), Sedentary Alzheimer's (AS), and Trained Alzheimer's (AT). Animals were submitted to stereotactic surgery and received a hippocampal injection of Aβ1-42 or a saline solution. Seven days after surgery, twelve days of treadmill adaptation followed by five maximal running tests (MRT) and fifty-five days of HIIT, rats underwent the Morris water maze test. The animals were then euthanized, and their gastrocnemius muscle tissue was extracted to analyze the Fibronectin type III domain containing 5 (FNDC5), PPARG Coactivator 1 Alpha (PPARGC1A), and Integrin subunit beta 5 (ITGB5-R) expression by qRT-PCR in addition to cross-sectional areas. The HIIT prevents the cognitive deficit induced by the infusion of amyloid β1-42 (p < 0.0001), causes adaptation of muscle fibers (p < 0.0001), modulates the gene expression of FNDC5 (p < 0.01), ITGB5 (p < 0.01) and PPARGC1A (p < 0.01), and induces an increase in peripheral protein expression of FNDC5 (p < 0.005). Thus, we conclude that HIIT can prevent cognitive damage induced by the infusion of Aβ1-42, constituting a non-pharmacological tool that modulates important genetic and protein pathways.

Angiotensin II-induced miR-31-5p upregulation promotes vascular smooth muscle cell proliferation and migration

Angiotensin II (Ang II) plays a central role in vascular smooth muscle cell (VSMC) proliferation and migration, being key to regulate vascular function and promote vascular remodeling in cardiovascular diseases. We recently showed that miR-31-5p promoted oxidative stress in spontaneously hypertensive rats. In this study, we aim to investigate whether miR-31-5p and fibronectin type III domain-containing 5 (FNDC5) contribute to Ang II-induced VSMC proliferation and migration. Experiments were performed in primary VSMCs of wide-type (WT) and FNDC5−/− mice as well as the rat A7r5 cell line. We found that Ang II increased miR-31-5p level, reduced FNDC5 expression and stimulated VSMC proliferation and migration, which were aggravated by miR-31-5p mimic, and prevented by miR-31-5p inhibitor in VSMCs. The Ang II-induced VSMC proliferation were prevented by exogenous FNDC5 in both WT and FNDC5−/− mice, while the effects were more significant in FNDC5−/− mice. Furthermore, exogenous FNDC5 reversed the effects of miR-31-5p mimic on VSMC proliferation and migration in Ang II-treated VSMCs. Meanwhile, FNDC5 deficiency prevented the effects of miR-31-5p inhibitor on VSMC proliferation and migration in Ang II-treated VSMCs. In conclusion, our findings demonstrate that the miR-31-5p upregulation and the following FNDC5 downregulation contribute to Ang II-induced VSMC proliferation and migration.

Pentamethylquercetin Regulates Lipid Metabolism by Modulating Skeletal Muscle-Adipose Tissue Crosstalk in Obese Mice.

Irisin is an exercise-induced hormone that regulates lipid metabolism. The present study investigates whether the anti-obesity effect of the natural flavonoid pentamethylquercetin (PMQ) is related to irisin secretion from skeletal muscle in whole animals and cultured cells. Obese mice induced by monosodium glutamate were administered oral PMQ to determine blood irisin level and in vivo parameters of lipid metabolism, and cultured mouse C2C12 myoblasts and 3T3-L1 preadipocytes were employed to investigate the related molecular identities. PMQ increased circulating irisin and decreased bodyweight, insulin, and lipid levels accompanied with increasing brown-like adipocyte formation in obese mice. The brown adipocyte marker uncoupling protein 1 (UCP-1) and other brown-like adipocyte-specific genes and/or markers were increased in mouse white fat tissue, while PMQ treatment reversed the above changes. PMQ also dose-dependently increased the reduced levels of AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and fibronectin type III domain-containing 5 (FNDC5) signal molecules in obese mice. Interestingly, the irisin level was increased in the culture medium of C2C12 cells treated with PMQ, and the conditioned medium stimulated the brown-like transition of 3T3-L1 preadipocytes with the increased expression of PGC-1α, FNDC5, UCP-1, and other brown-like adipocyte-specific genes. The effects of conditioned culture medium were abolished in C2C12 cells with silenced PGC-1α. On the other hand, PMQ-induced upregulation of PGC-1α and FNDC5 expression was reduced by AMPK inhibitor Compound C in C2C12 cells. Our results demonstrate the novel information that PMQ-induced irisin secretion from skeletal muscle involves the improvement of metabolic dysfunction in obese mice via activating the AMPK/PGC-1α/FNDC5 signal pathway, suggesting that PMQ modulates skeletal muscle-adipose tissue crosstalk and may be a promising drug candidate for treating obesity and obesity-related metabolic diseases.

Sestrin2 ablation attenuates the exercise-induced browning of white adipose tissue in C57BL/6J mice.

With exercise, white adipose tissues (WAT) are readily convertible to a "brown-like" state, altering from lipid-storing to energy-catabolizing function, which counteracts obesity and increases insulin sensitivity. Sestrin2 (SESN2) is a stress-inducible protein that can regulate the cold-induced increase of uncoupling protein 1 (UCP1), which is paramount for the thermogenic capacity of brown-like WAT. This study aimed to elucidate the necessity of SESN2 in mediating exercise-induced browning of WAT. We used 8-week, male wild-type and SESN2 knockout C57BL/6J mice to explore the potential role of SESN2 in the exercise-induced WAT browning process. Over a 3-week intervention (sedentary versus treadmill exercise, normal chow versus 60% high-fat diet), we examined the exercise-induced alterations of the browning phenotype in different depots of white fat. In vitro, 3T3-L1 pre-adipocytes and primary adipocytes were used to determine the potential mechanism. Our data revealed that SESN2 was required for the exercise-induced subcutaneous WAT (scWAT) browning. This may be mediated by higher fibronectin type III domain containing 5 (FNDC5) contents in scWAT locally, rather than skeletal muscle FNDC5 expression and circulating serum irisin levels. SESN2 ablation significantly impaired the exercise-improved glucose metabolism, where browning of scWAT may serve as an essential pathway. Moreover, SESN2 ablation significantly attenuated the exercise-promoted respiratory exchange ratio and indexes of energy metabolism (oxygen uptake and energy expenditure). Taken together, our results provided evidence that SESN2 is a key integrating factor in driving the diverse metabolic benefits conferred by aerobic exercise.

GLP-1 Induces the Expression of FNDC5 Derivatives That Execute Lipolytic Actions.

Multiple GLP-1-derived therapeutics are clinically used to treat type 2 diabetes and obesity. However, the underlying mechanism of how these drugs regulate the body weight of obese patients remains incompletely understood. Here, we report that the lipolysis effects of GLP-1 on β cells can depend on its induced expression of fibronectin type III domain containing 5 (FNDC5). The transmembrane FNDC5 is a precursor of the recently identified hormone irisin that possesses a range of bioactivities, including anti-obesity and anti-diabetes. We revealed that GLP-1 upregulates the expression and secretion of FNDC5 in β cells, while GLP-1 itself fails to activate the lipolysis genes in FNDC5-knockout β cells. In addition, liraglutide, a clinically used GLP-1 receptor agonist, induced the expression of FNDC5 in mouse pancreas and brain tissues and increased the serum level of secreted FNDC5. Furthermore, we observed the expression of the well-known membrane-associated FNDC5 and a novel, secretable FNDC5 (sFNDC5) isoform in β cells and multiple rat tissues. Recombinant sFNDC5 stimulated lipolysis of wild type and FNDC5-knockout β cells. This new isoform further induced lipolysis and browning of adipocytes, and similar to irisin, executed potent anti-obesity activities in an obese mouse model. Overall, our studies provided new mechanistic insights into GLP-1’s anti-obesity actions in which GLP-1 induces the secretion of FNDC5 derivatives from its responsive organs that then mediate its anti-obesity activities.

Ameliorative effects of garlic oil on FNDC5 and irisin sensitivity in liver of streptozotocin-induced diabetic rats.

This study was aimed to investigate the effects of garlic oil (GO), an important natural constituent used in alleviating diabetes and its complications, on the expression levels of irisin and related genes. Thirty-two rats were divided into four groups: Control, Diabetes-Control, Diabetes+GO 100 mg/kg/day and Control+GO 100 mg/kg/day for 45 days. The measurements included: changes in liver Peroxisome proliferator-activated receptor-gamma-coactivator (PGC)-1α, Fibronectin Type-III-Domain-Containing5 (FNDC5), irisin expression, mRNA expression of p38 and TNF-α (Tumour necrosis factor-α), total-antioxidant-status (L-TAS; S-TAS), total-oxidant-status (L-TOS; S-TOS) in liver and serum, respectively. There was a significant reduction in serum levels of irisin and S-TAS and expression of PGC-1α and FNDC5 in liver in Diabetes-control compared to Control-group, while a significant increase in serum levels of fasting blood glucose (FBG) and TOS, also p38 and TNF-α expressions in liver. In Diabetes+GO group, there was a significant increase in serum irisin and S-TAS, also expression of PGC-1α and FNDC5 in liver, while serum FBG, S-TOS levels, and mRNA expression of p38 and TNF-α in liver were decreased compared to Diabetes-control group (P < 0.05). GO alleviated the diabetic liver injury by decreasing Oxidative-Stress parameters and regulation PGC-lα, FNDC5, irisin and P38, keeping the balance of TAS/TOS and TNF-α.

Extracellular vesicle-mediated miR135a-5p transfer in hypertensive rat contributes to vascular smooth muscle cell proliferation via targeting FNDC5

BackgroundExtracellular vesicles (EVs) from vascular adventitial fibroblasts (AFs) contribute to the proliferation of vascular smooth muscle cells (VSMCs) and vascular remodeling in spontaneously hypertensive rat (SHR). This study shows the crucial roles of EVs-mediated miR135a-5p transfer in VSMC proliferation and the underlying mechanisms in hypertension.MethodsAFs and VSMCs were obtained from the aorta of Wistar-Kyoto rat (WKY) and SHR. EVs were isolated from the culture of AFs with ultracentrifugation method.ResultsMiR135a-5p level in SHR-EVs was significantly increased. MiR135a-5p inhibitor prevented the SHR-EVs-induced VSMC proliferation. Fibronectin type III domain containing 5 (FNDC5) was a target gene of miR135a-5p. FNDC5 level was lower in VSMCs of SHR. MiR135a-5p inhibitor not only increased FNDC5 expression, but reversed the SHR-EVs-induced FNDC5 downregulation in VSMCs of SHR. MiR135a-5p mimic inhibited FNDC5 expression, but failed to promote the SHR-EVs-induced FNDC5 downregulation in VSMCs of SHR. Exogenous FNDC5 prevented the SHR-EVs-induced VSMC proliferation of both WKY and SHR. Knockdown of miR135a-5p in fibroblasts completely prevented the upregulation of miR135a-5p in the EVs. The SHR-EVs from the miR135a-5p knockdown-treated fibroblasts lost their roles in inhibiting FNDC5 expression and promoting proliferation in VSMCs of both WKY and SHR.ConclusionsIncreased miR135a-5p in the SHR-EVs promoted VSMC proliferation of WKY and SHR via inhibiting FNDC5 expression. MiR135a-5p and FNDC5 are crucial targets for intervention of VSMC proliferation in hypertension.

Hepatic FNDC5 is a potential local protective factor against Non-Alcoholic Fatty Liver

The proteolytic cleavage of Fibronectin type III domain-containing 5 (FNDC5) generates soluble irisin. Initially described as being mainly produced in muscle during physical exercise, irisin mediates adipose tissue thermogenesis and also regulates carbohydrate and lipid metabolism. The aim of this study was to evaluate the hepatic expression of FNDC5 and its role in hepatocytes in Non-Alcoholic Fatty Liver (NAFL). Here we report that hepatic expression of FNDC5 increased with hepatic steatosis and liver injury without impacting the systemic level of irisin in mouse models of NAFLD (HFD and MCDD) and in obese patients. The increased Fndc5 expression in fatty liver resulted from its upregulation in hepatocytes and non-parenchymal cells in mice. The local production of Fndc5 in hepatocytes was influenced by genotoxic stress and p53-dependent pathways. The down-regulation of FNDC5 in human HepG2 cells and in primary mouse hepatocytes increased the expression of PEPCK, a key enzyme involved in gluconeogenesis associated with a decrease in the expression of master genes involved in the VLDL synthesis (CIDEB and APOB). These alterations in FNDC5-silenced cells resulted to increased steatosis and insulin resistance in response to oleic acid and N-acetyl glucosamine, respectively. The downregulation of Fndc5 also sensitized primary hepatocytes to apoptosis in response to TNFα, which has been associated with decreased hepatoprotective autophagic flux. In conclusion, our human and experimental data strongly suggest that the hepatic expression of FNDC5 increased with hepatic steatosis and its upregulation in hepatocytes could dampen the development of NAFLD by negatively regulating steatogenesis and hepatocyte death.

The saturated fatty acid palmitate induces insulin resistance through Smad3-mediated down-regulation of FNDC5 in myotubes

Elevated plasma free fatty acid (FFA) levels are associated with insulin resistance and can cause lipotoxicity in skeletal muscles. In response to FFAs, skeletal muscle can secrete a variety of cytokines. Irisin, one such muscle-secreted cytokine, can improve glucose tolerance, glucose uptake, and lipid metabolism. It is produced by the transmembrane protein fibronectin type Ⅲ domain containing 5 (FNDC5) by specific proteases. The purpose of this study was to investigate the regulatory mechanisms of the FNDC5 response to palmitate and their relationships with insulin resistance in C2C12 myotubes. RNA sequencing analysis results from C2C12 myotubes treated with palmitate showed that palmitate could activate the TGF-β signaling pathway. Palmitate directly affected the expression of Smad3, but not its phosphorylation level, in C2C12 myotubes. Furthermore, knockdown and knockout of Smad3 alleviated the inhibitory effect of palmitate on the expression of FNDC5. In contrast, overexpression of Smad3 aggravated the inhibition of FNDC5 expression. There is a Smad3 binding motif in the −660 bp to −649 bp region of the Fndc5 promoter. CRISPR/Cas9 knockout of this region also alleviated the inhibition of FNDC5 expression in response to palmitate. More importantly, inhibition of FNDC5 expression mediated by Smad3 led to a decrease in insulin sensitivity in C2C12 myotubes. Collectively, these findings suggest that palmitate could induce insulin resistance through Smad3-mediated down-regulation of the Fndc5 gene.

Effect of electroacupuncture combined with treadmill exercise on body weight and expression of PGC-1α， Irisin and AMPK in skeletal muscle of diet-induced obesity rats

To investigate the effect of electroacupuncture (EA) plus treadmill exercise on the expression of peroxisome proliferator activated receptor γ coactivator 1α(PGC-1α), Irisin, AMP-activated protein kinase (AMPK) in skeletal muscle of diet-induced obesity (DIO) rats, so as to explore its mechanism underlying body reduction promotion. Forty-two male SD rats were divided into normal diet (control, n＝10), high fat diet (model), EA, treadmill exercise and EA plus treadmill exercise (combination) groups (n＝8 in each of the latter 4 groups). The obesity model was established by feeding the rats with high fat diet. EA (2 Hz/15 Hz, 1 mA) was applied to bilateral "Zusanli" (ST36) and "Tianshu" (ST25) for 30 min, 5 times per week for a total of 8 weeks. Rats of the treadmill exercise group were forced to perform exercise on a treadmill (16 m/min) for 30 min, 5 times per week for a total of 8 weeks. Rats in the combination group received the above-mentioned two methods. During the treatment, rats in the control group were fed with normal fodder, rats in other groups were fed with high fat fodder, and their body weight was measured once a week. The expression levels of PGC-1α， fibronectin type Ⅲ domain containing 5 (FNDC5), AMPK mRNA and protein of skeletal muscle were measured by quantitative real-time PCR and Western blot，respectively. After modeling, the body weight was significantly increased (P<0.05), and the expression levels of PGC-1α and FNDC5 mRNA and protein, AMPK mRNA and phosphorylated AMPK (p-AMPK) protein in the skeletal muscle were considerably decreased in the model group relevant to the control group (P<0.05). Following the treatment, the body weight was significantly down-regulated, while the expression levels of PGC-1α and FNDC5 mRNAs and proteins, AMPK mRNA and p-AMPK protein were obviously up-regulated in the EA, treadmill exercise and combination groups relevant to the model group (P<0.05). The therapeutic effect of EA plus treadmill exercise was significantly superior to those of both simple EA and simple treadmill exercise in down-regulating the body weight, as well as in up-regulating the expression of PGC-1α and FNDC5 mRNAs and proteins, AMPK mRNA, and p-AMPK protein (P<0.05). Both EA and treadmill exercise can significantly increase the expression of PGC-1α， FNDC5 and p-AMPK in skeletal muscle of DIO rats, suggesting their efficacy in restoring fatty acid oxidation in skeletal muscle cells and improving mitochondrial function, which may contribute to their function in body reduction. The therapeutic effect of EA plus treadmill exercise is better than that of simple EA and simple treadmill exercise.

FNDC5 alleviates oxidative stress and cardiomyocyte apoptosis in doxorubicin-induced cardiotoxicity via activating AKT

Oxidative stress and cardiomyocyte apoptosis play critical roles in doxorubicin (DOX)-induced cardiotoxicity. Previous studies indicated that fibronectin type III domain-containing 5 (FNDC5) and its cleaved form, irisin, could preserve mitochondrial function and attenuate oxidative damage as well as cell apoptosis, however, its role in DOX-induced cardiotoxicity remains unknown. Our present study aimed to investigate the role and underlying mechanism of FNDC5 on oxidative stress and cardiomyocyte apoptosis in DOX-induced cardiotoxicity. Cardiomyocyte-specific FNDC5 overexpression was achieved using an adeno-associated virus system, and then the mice were exposed to a single intraperitoneal injection of DOX (15 mg/kg) to generate DOX-induced cardiotoxicity. Herein, we found that FNDC5 expression was downregulated in DOX-treated murine hearts and cardiomyocytes. Fndc5 deficiency resulted in increased oxidative damage and apoptosis in H9C2 cells under basal conditions, imitating the phenotype of DOX-induced cardiomyopathy in vitro, conversely, FNDC5 overexpression or irisin treatment alleviated DOX-induced oxidative stress and cardiomyocyte apoptosis in vivo and in vitro. Mechanistically, we identified that FNDC5/Irisin activated AKT/mTOR signaling and decreased DOX-induced cardiomyocyte apoptosis, and moreover, we provided direct evidence that the anti-oxidant effect of FNDC5/Irisin was mediated by the AKT/GSK3β/FYN/Nrf2 axis in an mTOR-independent manner. And we also demonstrated that heat shock protein 20 was responsible for the activation of AKT caused by FNDC5/Irisin. In line with the data in acute model, we also found that FNDC5/Irisin exerted beneficial effects in chronic model of DOX-induced cardiotoxicity (5 mg/kg, i.p., once a week for three times, the total cumulative dose is 15 mg/kg) in mice. Based on these findings, we supposed that FNDC5/Irisin was a potential therapeutic agent against DOX-induced cardiotoxicity.

Icariin induces irisin/FNDC5 expression in C2C12 cells via the AMPK pathway

Backgroud Icariin, a major bioactive pharmaceutical component of the Chinese herbal medicine Epimedii Herba, has demonstrated lipid-lowering and anti-obesity effects. Irisin/ fibronectin type III domain-containing 5 (FNDC5) protects against obesity by inducing browning in white adipose tissue. Objectives This study investigated the effects of icariin on irisin/FNDC5 expression in C2C12 myotubes. Method Cultured murine C2C12 myocytes were used to study the effects of icariin on irisin/FNDC5 expressions by Western-blot, qPCR, Elisa and Immunofluorescence. We also investigated FNDC5 expression in icariin-treated intact mice. Results Icariin increased irisin/FNDC5 protein levels. mRNA levels of irisin/FNDC5 were also increased in C2C12 myocytes after treatment with icariin. Icariin increased peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1α) protein and mRNA levels. Additionally, icariin exposure resulted in phosphorylation of AMP-activated protein kinase (AMPK) in a dose-dependent manner. The regulatory effect of icariin on FNDC5 protein expression was blocked by the AMPK antagonist compound C or silencing of AMPK, suggesting that icariin increased FNDC5 protein expression via the AMPK pathway. In vivo, icariin decreased body weight gain in C57BL/6 mice and increased FNDC5, PGC-1α, and p-AMPK expression levels in skeletal muscle. Conclusions Taken together, our results indicated that icariin induces irisin/FNDC5 expression via the AMPK pathway, indicating that icariin may be promising as an anti-obesity drug.

Urotensin II Induces Mice Skeletal Muscle Atrophy Associated with Enhanced Autophagy and Inhibited Irisin Precursor (Fibronectin Type III Domain Containing 5) Expression in Chronic Renal Failure

Background/Aims: Skeletal muscle atrophy is one of the main manifestations of protein energy wasting. We hypothesized that urotensin II (UII) can lead to skeletal muscle atrophy through upregulating autophagy and affecting Irisin precursor fibronectin type III domain containing 5 (FNDC5) expressions. Methods: Three animal models (the sham operation, wild-type C57BL/6 mice with 5/6 nephrectomy, UII receptor (UT) gene knockout (UTKO) mice with 5/6 nephrectomy) were designed. Skeletal muscle weight, cross-sectional area (CSA) along with UII, FNDC5, LC3, and p62 expression were investigated. C2C12 cells were differentiated for up to 4 days into myotubes. These cells were then exposed to different UII concentrations (10^–5 to 10^–7 M) for 6–12 h and analyzed for the expressions of autophagic markers. These cells were also exposed to the same predetermined UII concentrations for 48–72 h and analyzed for the FNDC5 expression. Myotube diameter was measured. Results: Upregulation of UII expression in skeletal muscle tissue was accompanied by reduced muscle weight and skeletal muscle CSA in the 2 posterior limbs, upregulated autophagy markers expression, and downregulated FNDC5 expression in 5/6 nephrectomy mice. The decrease of skeletal muscle weight, skeletal muscle CSA, downregulation of FNDC5 expression, and the upregulation of autophagy markers were inhibited in UTKO with 5/6 nephrectomy mice. Our in vitrostudy showed that UII could directly decrease myotube diameter, induce autophagy markers upregulation, and inhibit expression of FNDC5. When UII receptor gene was interfered by UT-specific siRNA, UII induced autophagy markers upregulation and FNDC5 downregulation were inhibited. Conclusion: We are the first to verify UII induces mice skeletal muscle atrophy associated with enhanced skeletal muscle autophagy and inhibited FNDC5 expression in chronic renal failure.

Irisin and Fibronectin Type III Domain-Containing 5 Responses to Exercise in Different Environmental Conditions

Fibronectin type III domain-containing 5 (FNDC5) is a skeletal muscle membrane-bound precursor to the myokine irisin. Irisin is involved in stimulating adipose tissue to become more metabolically active in order to produce heat. It is unknown how exercising in different temperature environments affects the response of FNDC5 gene expression and blood irisin concentration. PURPOSE: To determine the effects of exercise in a hot (33 °C), cold (7 °C), and room temperature (RT, 20 °C) environment on the skeletal muscle gene expression of FNDC5 and the blood concentrations of irisin. METHODS: Twelve recreationally trained males completed three separate, 1 h cycling bouts at 60% of Wmax in a hot, cold, and RT environment followed by three hours of recovery at room temperature. Blood samples were taken from the antecubital vein and muscle biopsies were taken from the vastus lateralis pre-, post-, and 3 h post-exercise. RESULTS: Plasma concentrations of irisin did not change from pre- (9.23 ± 2.68 pg · mL-1) to post-exercise (9.6 ± 0.2 pg · mL-1, p = 0.068), but decreased from post-exercise to 3 h post-exercise (8.9 ± 0.5 pg · mL-1, p = 0.047), regardless of temperature. However, when plasma volume shifts were considered, no differences were found in irisin at pre-, post-, or 3 h post- exercise (p = 0.086). There were no differences between trials for irisin plasma concentrations (p = 0.984). No differences in FNDC5 mRNA were observed between the hot, cold, and RT trials or between pre-, post-, and 3 h post-exercise time points (p > 0.05). CONCLUSION: These data indicate that the temperature in which exercise takes place does not influence FNDC5 skeletal muscle transcription or circulating irisin in a human model. Funding provided by the University of Nebraska at Omaha Graduate Research and Creative Activity Grant and the National Institute for General Medical Science (NIGMS, 5P20GM103427).

Glucocorticoid receptor positively regulates transcription of FNDC5 in the liver

Irisin is secreted by skeletal muscle during exercise and influences energy and metabolic homeostasis. This hormone is a cleaved and secreted fragment of fibronectin type III domain-containing 5 (FNDC5). Elucidation of the FNDC5 gene regulation mechanism is necessary to clarify the function of irisin as a potential therapeutic target in human metabolic diseases. Thus, we investigated the genetic and epigenetic mechanisms that regulate expression of the FNDC5 gene. FNDC5 mRNA was strong expressed in major energy-dependent human tissues, including heart, brain, liver, and skeletal muscle. Promoter analysis of the FNDC5 gene revealed that the core promoter region of the FNDC5 gene contained one CpG island that was located just upstream of the transcriptional start site for variants 2 and 3. Treatment with the histone deacetylase inhibitor sodium butyrate and the demethylating agent 5-azacytidine increased mRNA expression of FNDC5 in Huh7 cells. Prediction of transcription factor binding sites suggested that the glucocorticoid receptor was involved in the regulation of FNDC5 expression, and indeed, cortisol treatment increased mRNA expression of FNDC5 in Huh7 cells. Collectively, these findings offer insight into the genetic and epigenetic regulation of FNDC5, providing the initial steps required for understanding the role of irisin in the metabolic homeostasis.

Effects of Dietary Macronutrient Composition on FNDC5 and Irisin in Mice Skeletal Muscle.

Fibronectin type III domain containing 5 (FNDC5) and its protein product Irisin are therapeutic targets for obesity-associated disorders. Irisin plays an important role in energy regulation, inducing browning of white adipocytes, and improving obesity. We aimed to investigate the association between muscle Irisin expression and dietary quality. Twenty-eight female mice were divided into four groups and fed the following experimental diets for 60 days: standard diet (SD), high-carbohydrate diet (HCD), high-fat diet (HFD), and high-protein diet (HPD). We evaluated body weight, food intake, serum total cholesterol, triacylglycerol, and glucose. We also performed glucose tolerance and insulin sensitivity tests. Expression of FNDC5 was evaluated by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) of soleus muscle. Western blot was used to assess Irisin protein expression. The major finding of the present study was that HFD and HCD were associated with a downregulation of FNDC5. In addition to these results, we noted a significant reduction in skeletal muscle Irisin level. HPD prevented reductions of both FNDC5 and Irisin levels, as well as increased brown adipose tissue, compared to the control group. In conclusion, we observed that the HPD type of diet can change both FNDC5 expression and Irisin levels. Thus, the HPD might be the most appropriate diet to achieve high amounts of Irisin, a target molecule for the treatment of obesity and its co-morbidities.

Fibronectin type III domain containing 5 expression in skeletal muscle in chronic heart failure-relevance of inflammatory cytokines.

BackgroundChronic heart failure (CHF) is commonly associated with muscle atrophy and increased inflammation. Irisin, a myokine proteolytically processed by the fibronectin type III domain containing 5 (FNDC5) gene and suggested to be Peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α activated, modulates the browning of adipocytes and is related to muscle mass. Therefore, we investigated whether skeletal muscle FNDC5 expression in CHF was reduced and if this was mediated by inflammatory cytokines and/or angiotensin II (Ang-II).MethodsSkeletal muscle FNDC5 mRNA/protein and PGC-1α mRNA expression (arbitrary units) were analysed in: (i) rats with ischemic cardiomyopathy; (ii) mice injected with tumour necrosis factor-α (TNF-α) (24 h); (iii) mice infused with Ang-II (4 weeks); and (iv) C2C12 myotubes exposed to recombinant cytokines or Ang-II. Circulating TNF-α, Ang-II, and irisin was measured by ELISA.ResultsIschemic cardiomyopathy reduced significantly FNDC5 protein (1.3 ± 0.2 vs. 0.5 ± 0.1) and PGC-1α mRNA expression (8.2 ± 1.5 vs. 4.7 ± 0.7). In vivo TNF-α and Ang-II reduced FNDC5 protein expression by 28% and 45%, respectively. Incubation of myotubes with TNF-α, interleukin-1ß, or TNF-α/interleukin-1ß reduced FNDC5 protein expression by 47%, 37%, or 57%, respectively, whereas Ang-II had no effect. PGC-1α was linearly correlated to FNDC5 in all conditions. In CHF, animals circulating TNF-α and Ang-II were significantly increased, whereas irisin was significantly reduced. A negative correlation between circulating TNF-α and irisin was evident.ConclusionA reduced expression of skeletal muscle FNDC5 in ischemic cardiomyopathy is likely modulated by inflammatory cytokines and/or Ang-II via the down-regulation of PGC-1α. This may act as a protective mechanism either by slowing the browning of adipocytes and preserving energy homeostasis or by regulating muscle atrophy.

Effect of obesity and exercise on the expression of the novel myokines, Myonectin and Fibronectin type III domain containing 5.

Metabolic dysfunction in skeletal muscle is a major contributor to the development of type 2 diabetes. Endurance exercise training has long been established as an effective means to directly restore skeletal muscle glucose and lipid uptake and metabolism. However, in addition to the direct effects of skeletal muscle on glucose and lipids, there is renewed interest in the ability of skeletal muscle to coordinate metabolic activity of other tissues, such as adipose tissue and liver. The purpose of this study was to examine the effects of endurance exercise on the expression level of two novel muscle-derived secreted factors, or myokines, Myonectin and Fibronectin type III domain containing 5 (FNDC5), the precursor for Irisin.Methods. We performed immunoblot analysis and quantitative real-time PCR analysis of Myonectin and FNDC5 in the diaphragm muscles of obese Zucker rat (OZR) and lean Zucker rat (LZR) with 9 weeks of aerobic training on a motorized treadmill.Results. We show that myonectin gene expression is increased in the OZR model of obesity and decreases with exercise in both lean and obese Zucker rats. Conversely, myonectin protein concentration was elevated with exercise. Similarly, FNDC5 mRNA levels are significantly higher in the OZR, however exercise training had no effect on the expression level of FNDC5 in either the LZR or OZR. We did not observe any difference in muscle protein content of Irisin with obesity or exercise.Conclusion. Our data shows that exercise training does not increase either FNDC5 or myonectin gene expression, indicating that increased transcriptional regulation of these myokines is not induced by exercise. However, our data also indicates a yet to be explored disconnect between myonectin gene expression and protein content. Further, this report highlights the importance of verifying reference genes when completing gene expression analysis. We found that many commonly used reference genes varied significantly by obesity and/or exercise and would have skewed the results of this study if used to normalize gene expression data. The unstable reference genes include: beta-Actin, beta-2-microglobulin, Non-POU domain containing, octamer-binding, Peptidylprolyl isomerase H, 18S ribosomal RNA, TATA box binding protein and Transferrin receptor.