Julia Brenmoehl

Irisin - a myth rather than an exercise-inducible myokine.

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.

Dual control of mitochondrial biogenesis by sirtuin 1 and sirtuin 3

In this review, we discuss the dual control of mitochondrial biogenesis and energy metabolism by silent information regulator-1 and -3 (SIRT1 and SIRT3). SIRT1 activates the peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α)-mediated transcription of nuclear and mitochondrial genes encoding for proteins promoting mitochondria proliferation, oxidative phosphorylation and energy production, whereas SIRT3 directly acts as an activator of proteins important for oxidative phosphorylation, tricarboxylic acid (TCA) cycle and fatty-acid oxidation and indirectly of PGC-1α and AMP-activated protein kinase (AMPK). The complex network involves different cellular compartments, transcriptional activation, post-translational modification and a plethora of secondary effectors. Overall, the mode of interaction between both sirtuin family members may be considered as a prominent case of molecular job-sharing.

Irisin Is Elevated in Skeletal Muscle and Serum of Mice Immediately after Acute Exercise

Recent findings regarding the response of fibronectin type III domain-containing protein 5 (Fndc5) and irisin to exercise are partly controversial. While the 25 kDa form of Fndc5 can be observed in muscle and serum of different species, the ~12 kDa irisin band was not detectable up to now. The present study aimed to clarify whether irisin exists in its theoretical size of ~12 kDa in mice and if it is affected by exercise. Male mice were randomly assigned to a sedentary control group (CO), a group with free access to running wheels (RW), and a treadmill group (TM). Blood and leg muscles were collected to investigate the regulatory cascade including peroxisome proliferator-activated receptor gamma co-activator 1-alpha (Ppargc1a) and Fndc5. In western blot analysis, antibodies were used capable of differentiation between full-length Fndc5 and irisin. This enabled us to demonstrate that irisin exists in muscle and serum of mice independent of exercise and that it is increased immediately after acute exercise. Different transcripts of Ppargc1a mRNA, but not Fndc5 mRNA, were up-regulated in the TM group. Furthermore, neither Fndc5 (25 kDa) nor Ppargc1a protein was elevated in muscle tissue. The Ppargc1a-Fndc5/irisin pathway did not clearly respond to mild exercise in the RW group. Our results provide evidence for the existence of irisin and for its immediate response to acute exercise in mice.

Locus Characterization and Gene Expression of Bovine FNDC5: Is the Myokine Irisin Relevant in Cattle?

The transmembrane protein FNDC5 was recently characterized as precursor of an exercise induced myokine named irisin. Previous studies found a relationship between circulating irisin levels and muscle mass in humans. Consequently, we tested the hypothesis whether FNDC5/irisin is involved in the modulation of body composition in cattle. Since information on the bovine FNDC5 locus was scarce, we characterized the gene experimentally as prerequisite for these investigations. We provide here a revised and extended gene model for bovine FNDC5. Although similarly organized like the human and murine loci, a higher variability was observed at transcript level in the bovine locus. FNDC5 mRNA was abundant in bovine skeletal muscle and was detected at lower levels in adipose tissue and liver. There were no expression differences between two groups of bulls highly different in muscularity and adiposity. Full-length FNDC5 protein (25 kDa) was present in bovine skeletal muscle independent of muscularity. Neither FNDC5 nor its putatively secreted peptide irisin were found in circulation of bulls. In contrast, we demonstrated that FNDC5 (25 kDa) and irisin (12 kDa) were present in murine skeletal muscle and that irisin was circulating in murine serum. This indicates fundamental differences in the regulation of FNDC5 and irisin between rodents and cattle.

Metabolic adaptations in the liver of born long-distance running mice.

PURPOSE Long-distance runners have increased needs of energy supply. To unravel genetically based mechanisms required for efficient energy supply, we have analyzed hepatic metabolism of mice characterized by the inborn capacity to perform as long-distance runners. METHODS The mouse model had been established by phenotypic selection for high treadmill performance for 90 generations and was characterized by approximately 3.8-fold higher running capacities (Dummerstorf high Treadmill Performance mouse line [DUhTP]) compared with unselected and also untrained controls (Dummerstorf Control mouse line [DUC]). From 7-wk-old male mice, serum and liver samples were collected and analyzed for messenger RNA, protein, and metabolite levels, respectively. RESULTS In livers from DUhTP mice, we identified significantly higher messenger RNA transcript levels of peroxisome proliferator-activated receptor delta and higher protein levels of sirtuin-1, acetyl-CoA-synthetase, acetyl-CoA-carboxylase, phosphoenolpyruvate carboxykinase, and glutamate-dehydrogenase, suggesting higher gluconeogenesis and lipogenesis in DUhTP mice. In fact, higher hepatic levels of glycogen and triglycerides as well as higher concentrations of carbohydrate, fatty acid, and cholesterol metabolites were found in DUhTP mice. In parallel, in DUhTP mice, which did not have access to running wheels, a marked hyperlipidemia (cholesterol = 160% ± 8%, triglycerides = 174% ± 14% of controls, respectively), and abdominal obesity (DUhTP = 0.396 ± 0.019 g, DUC = 0.291 ± 0.019 g) were found. CONCLUSIONS From our data, we conclude that the physiological basis of genetically fixed higher endurance-running performance in DUhTP marathon mouse is related to increased hepatic gluconeogenesis and lipogenesis. Expression of sirtuin 1 as well as of gluconeogenic and lipogenic key enzymes may be related to peroxisome proliferator-activated receptor delta. Metabolic adaptations presented in our study represent inborn features of superior endurance-running performance.

The RGD sequence present in IGFBP-2 is required for reduced glucose clearance after oral glucose administration in female transgenic mice

Recent studies suggest that insulin-like growth factor-binding protein-2 (IGFBP-2) affects both growth and metabolism. Whereas negative growth effects are primarily due to negative interference with IGF-I, the mechanisms for metabolic interference of IGFBP-2 are less clear. As we demonstrate, overexpression of IGFBP-2 in transgenic mice is correlated with a decelerated clearance of blood glucose after oral administration. IGFBP-2 carries an integrin-binding domain (RGD motif), which has been shown to also mediate IGF-independent effects. We thus asked if higher serum levels of IGFBP-2 without an intact RGD motif would also partially block blood glucose clearance after oral glucose application. In fact, transgenic mice overexpressing mutated IGFBP-2 with higher levels of IGFBP-2 carrying an RGE motif instead of an RGD were not characterized by decelerated glucose clearance. Impaired glucose tolerance was correlated with lower levels of GLUT4 present in plasma membranes isolated from muscle tissues after glucose challenge. At the same time, activation of TBC1D1 was depressed in mice overexpressing wild-type but not mutated IGFBP-2. Although we do not have reason to assume altered activation of IGF-I receptor or PDK1/Akt activation in both models, we have identified increased levels of integrin-linked kinase and focal adhesion kinase dependent on the presence of the RGD motif. From our results we conclude that impaired glucose clearance in female IGFBP-2 transgenic mice is dependent on the presence of the RGD motif and that translocation of GLUT4 in the muscle may be regulated by IGFBP-2 via RGD-dependent mechanisms.

Control of IGFBP-2 Expression by Steroids and Peptide Hormones in Vertebrates

IGFBP-2 (1) has been described as a brain tumor oncogene (2) and is widely expressed in cancers from different origins (3–8). IGFBP-2 alone cannot cause malignant transformation, yet progression of brain tumors to higher grade (9) and also has been provided as a protective element in earlier stages of multistage colon carcinogenesis (10). Therefore, it is crucial to understand the factors that determine expression patterns of IGFBP-2 under normal and malignant conditions. The present review provides a comprehensive update of known factors that have an impact on expression of IGFBP-2.

Bioanalytical validation for simultaneous quantification of non-aromatic steroids in follicular fluid from cattle via ESI-LC–MS/MS

The family of steroid hormones is quite attractive for the approach of phenotype monitoring in farm animals. Therefore, we developed a new protocol for the quantitative analysis of natural steroids in follicular fluid from dairy cows. The corresponding steroid profile, which consists of progesterone, corticosterone, hydrocortisone, testosterone, and androstenedione covering three distinct steroid classes, was determined by LC/MS. Quantification is achieved by use of steroid standards diluted in steroid-free follicular fluid as calibrators. Thus, the new protocol does not require deuterated standards. In order to correct for conditional performance of the analytical system we have used dexamethasone as an internal standard. The method was validated according to EMA guidelines. Within- and between-day variations were below 20% for most parameters assessed. All steroids assessed had lower limits of quantification in the range of 2.1 to 4.4ng/ml. We have established a simple and sensitive analytical system in order to step towards a broader and cost-efficient phenotyping analysis in follicular fluid from dairy cows.

Donor Nucleotide-Binding Oligomerization–Containing Protein 2 (NOD2) Single Nucleotide Polymorphism 13 Is Associated with Septic Shock after Allogeneic Stem Cell Transplantation

Single nucleotide polymorphisms (SNPs) within nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and toll-like receptor (TLR) 5 genes have been recently associated with the incidence and outcome of infections. In this study, we analyzed 38 patients with septic shock after allogeneic stem cell transplantation (allo-SCT) for an association of SNPs within NOD2 and TLR5 genes, with susceptibility to septic shock. One hundred twenty-seven transplant recipients unaffected by any infectious complications were used as controls. We found a significant association between the presence of donor NOD2 SNP13 (3016_3017insC) and the incidence of septic shock (P = .002). In multivariate analysis, donor NOD2 SNP13 appeared as an independent risk factor for the incidence of septic shock after allo-SCT. No association was found for recipient SNPs (NOD2 and TLR5) and donor NOD2 SNP8, SNP12, and TLR5-Stop SNP. Our results suggest that NOD2 SNP13 has an impact on the pathophysiology of severe infectious complications and is an independent risk factor for the development of septic shock after allo-SCT.

Skeletal muscle-specific overexpression of IGFBP-2 promotes a slower muscle phenotype in healthy but not dystrophic mdx mice and does not affect the dystrophic pathology

OBJECTIVE The insulin-like growth factor binding proteins (IGFBPs) are thought to modulate cell size and homeostasis via IGF-I-dependent and -independent pathways. There is a considerable dearth of information regarding the function of IGFBPs in skeletal muscle, particularly their role in the pathophysiology of Duchenne muscular dystrophy (DMD). In this study we tested the hypothesis that intramuscular IGFBP-2 overexpression would ameliorate the pathology in mdx dystrophic mice. DESIGN 4week old male C57Bl/10 and mdx mice received a single intramuscular injection of AAV6-empty or AAV6-IGFBP-2 vector into the tibialis anterior muscle. At 8weeks post-injection the effect of IGFBP-2 overexpression on the structure and function of the injected muscle was assessed. RESULTS AAV6-mediated IGFBP-2 overexpression in the tibialis anterior (TA) muscles of 4-week-old C57BL/10 and mdx mice reduced the mass of injected muscle after 8weeks, inducing a slower muscle phenotype in C57BL/10 but not mdx mice. Analysis of inflammatory and fibrotic gene expression revealed no changes between control and IGFBP-2 injected muscles in dystrophic (mdx) mice. CONCLUSIONS Together these results indicate that the IGFBP-2-induced promotion of a slower muscle phenotype is impaired in muscles of dystrophin-deficient mdx mice, which contributes to the inability of IGFBP-2 to ameliorate the dystrophic pathology. The findings implicate the dystrophin-glycoprotein complex (DGC) in the signaling required for this adaptation.