Steffen Maak

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.

Structural and functional characteristics of muscle fibres in pigs with different malignant hyperthermia susceptibility (MHS) and different meat quality

In pigs, intensive growth of the musculature is often accompanied by malignant hyperthermia susceptibility (MHS; n gene) and poorer meat quality. Using histological and histochemical methods, different fibre characteristics in the Longissimus muscle were found in Pietrain×German Landrace pigs with this gene defect. Compared to MHS homozygous negative pigs, groups with the n gene had increased diameters of the mean fibre types and increased glycolytic metabolic potential, as shown by a higher frequency of the fast twitch glycolytic type and a lower frequency of the slow twitch oxidative fibre type. Differences between the groups were also found in the number of angular and giant fibre types. Furthermore, a positive correlation was found between the frequency of oxidative fibres and the relative enzyme activity of NADH tetrazolium reductase. The changes correlated with lower pH and higher drip loss in meat from the MHS homozygous positive group. In conclusion, the different muscle fibre characteristics can be interpreted as endogenous factors which influence the physiological condition in the muscle of the live animal and meat quality post mortem.

Cellular conditions for intramuscular fat deposition in Japanese Black and Holstein steers

The experiment was conducted to study the development of intramuscular fat in Japanese Black (JB) compared to Holstein (HS) steers and to find breed differences for fat depot development and distribution in the carcass under equal feeding conditions. Additional to slaughter samples, biopsy samples of longissimus muscle (LM) and subcutaneous fat, taken at 10, 14, 18, and 22 months of age, were used for histological and molecular investigations. Japanese Black steers stored about 14% more fat in the LM (P = 0.001), resulting in larger marbling flecks (P < 0.001). Muscle fibers and intramuscular adipocytes in both breeds responded to the high energy feeding with significant enlargement, which was faster in JB. Histograms of intramuscular adipocytes size showed a shift toward larger cells during growth, but also the abundance of small, developing adipocytes. This development was accompanied by a correlated up-regulation of adipogenic genes until 22 months of age.

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.

Genetic spatial structure of European common hamsters (Cricetus cricetus) — a result of repeated range expansion and demographic bottlenecks

The spatial genetic structure of common hamsters (Cricetus cricetus) was investigated using three partial mitochondrial (mt) genes and 11 nuclear microsatellite loci. All marker systems revealed significant population differentiation across Europe. Hamsters in central and western Europe belong largely to two allopatric mitochondrial lineages south and northwest of the Carpathian and Sudetes. The southern group, ‘Pannonia’, comprises populations inside the Carpathian basin (Czech Republic, Hungary) while the second group, ‘North’, includes hamsters from Belgium, the Netherlands, France, and Germany. Isolation of the lineages is maintained by a combination of geographical and ecological barriers. Both main phylogeographical groups show signs of further subdivision. North is separated into highly polymorphic central German and less polymorphic western populations, which most likely split during late glacial expansion (15 000–10 000 bp). Clock estimates based on haplotype distributions predict a divergence of the two major lineages 85 000–147 000 bp. Expansion times fall during the last glaciation (115 000–10 000 bp) corroborating fossil data, which identify Cricetus cricetus as characteristic of colder climatic phases. Despite the allopatry of mt haplotypes, there is an overlap of nuclear microsatellite alleles between phylogeographical units. Although there are strong evidence that Pannonian hamsters have persisted inside the Carpathian basin over the last 50 000 years, genetic differentiation among European hamsters has mainly been caused by immigration from different eastern refugia. Possible source populations are likely to be found in the Ukrainian and the southern Russian plains — core areas of hamster distribution. From there, hamsters have repeatedly expanded during the Quaternary.

Multiple bottlenecks in threatened western European populations of the common hamster Cricetus cricetus (L.)

Common hamsters Cricetus cricetus (L.)show a highly fragmented distributionpattern across Europe. Over the last decades,human influence caused significant populationdeclines in particular at the western rangeboundary. Despite the initiation of breedingand release programs the genetic structure anddiversity of European common hamsterpopulations is largely unknown. In this study,hamsters from ten localities in five Europeancountries were investigated. Mitochondrialcontrol region was sequenced from 145 animalsrepresenting all sampled populations. 385hamster were screened for polymorphisms at 11microsatellite loci. Both marker systemsrevealed extensive genetic differentiationamong European common hamsters. Westernpopulations displayed very low levels of mtDNAdiversity (H = 0 − 0.2, Alsace, Limburg,Flanders, Baden-Wuerttemberg) compared toeastern populations from Saxony-Anhalt,Thuringia and Southern Moravia (H = 0.663− 0.816). Microsatellite analyses revealed asimilar pattern with low to moderate diversityvalues in western hamsters (A = 1.636 −5.364; He = 0.111 − 0.504) and highlevels of polymorphism in eastern hamsters(A = 8.909 − 9.818; He = 0.712− 0.786). High microsatellite based FSTmeasures (up to 0.635) suggest a typical islandmodel of distribution with no current gene flowbetween most areas. Western hamster populationsexhibit obvious similarities in mitochondrialhaplotype and microsatellite alleledistributions. Gene trees group westernhamsters consistently together on the samebranch but bootstrap values never reachedsignificance. There are strong indications thatlow diversity in western populations ispartially caused by a joint historic founderevent and not only by recent population breakdowns. Overlapping mitochondrial haplotypesprove a close association between westernhamsters and animals from the east German rangein the recent past which does not support theexistence of a separate subspecies C. c.canescens in Europe. Hamsters from southernMoravia emerged as the genetically mostdistinguished population and could be part of a different genetic lineage in Europe.

Agouti revisited: transcript quantification of the ASIP gene in bovine tissues related to protein expression and localization.

Beside its role in melanogenesis, the agouti signaling protein (ASIP) has been related to obesity. The potentially crucial role in adipocyte development makes it a tempting candidate for economic relevant, fat related traits in farm animals. The objective of our study was to characterize the mRNA expression of different ASIP transcripts and of putative targets in different bovine tissues, as well as to study consequences on protein abundance and localization. ASIP mRNA abundance was determined by RT-qPCR in adipose and further tissues of cattle representing different breeds and crosses. ASIP mRNA was up-regulated more than 9-fold in intramuscular fat of Japanese Black cattle compared to Holstein (p<0.001). Further analyses revealed that a transposon-derived transcript was solely responsible for the increased ASIP mRNA abundance. This transcript was observed in single individuals of different breeds indicating a wide spread occurrence of this insertion at the ASIP locus in cattle. The protein was detected in different adipose tissues, skin, lung and liver, but not in skeletal muscle by Western blot with a bovine-specific ASIP antibody. However, the protein abundance was not related to the observed ASIP mRNA over-expression. Immuno-histochemical analyses revealed a putative nuclear localization of ASIP additionally to the expected cytosolic signal in different cell types. The expression of melanocortin receptors (MCR) 1 to 5 as potential targets for ASIP was analyzed by RT-PCR in subcutaneous fat. Only MC1R and MC4R were detected indicating a similar receptor expression like in human adipose tissue. Our results provide evidence for a widespread expression of ASIP in bovine tissues at mRNA and, for the first time, at protein level. ASIP protein is detectable in adipocytes as well as in further cells of adipose tissue. We generated a basis for a more detailed investigation of ASIP function in peripheral tissues of various mammalian species.

Molecular heterogeneities of adipose depots - potential effects on adipose-muscle cross-talk in humans, mice and farm animals.

Adipose tissue is considered as a major endocrine organ that secretes numerous proteins called adipokines. The heterogeneous nature of adipose tissue in different parts of the body suggests respective heterogeneity of proteomes and secretomes. This review consolidates knowledge from recent studies targeting the diversity of different adipose depots affecting the pattern of secreted adipokines and discusses potential consequences for the cross-talk between adipose and skeletal muscle in humans, rodent models and farm animals. Special attention is paid to muscle-associated fat depots like inter- and intramuscular fat that become focus of attention in the context of the rather new notion of skeletal muscle as a major endocrine organ. Understanding the complexity of communication between adipocytes and skeletal muscle cells will allow developing strategies for improvement of human health and for sustainable production of high quality meat.

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.

Technical note: Determination of cell-specific gene expression in bovine skeletal muscle tissue using laser microdissection and reverse-transcription quantitative polymerase chain reaction1

Skeletal muscle is a very heterogeneous tissue consisting of diverse cell types with specific transcription profiles. Therefore, the measured mRNA abundance of a certain cell type marker is influenced by the transcriptional activity as well as by the usually unknown number of contributing cells in the sample. In studies on the transcriptional activity of adipogenic genes, as indicators for the development of intramuscular adipocytes, an altered number of adipocytes or respective progenitor cells can mask changes in transcriptional activity. To overcome this problem, we started to use laser microdissection to isolate RNA of adipocytes and muscle fibers separately for downstream analysis. Even muscle fiber types can be collected and analyzed separately. Laser microdissection in combination with biopsy techniques enables gene expression studies of particular cell types during the life cycle of an animal. First experiences using laser microdissection for adipogenic gene expression studies in bovine skeletal muscle are described, and the influence of sample preparation and future challenges are discussed.