Martina Langhammer

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.

Effect of a high-protein diet on food intake and liver metabolism during pregnancy, lactation and after weaning in mice.

Major hepatic metabolic pathways are involved in the control of food intake but how dietary proteins affect global metabolism to adjust food intake is incompletely understood, particularly under physiological challenging conditions such as lactation. In order to identify these molecular events, mice were fed a high‐protein (HP) diet from pregnancy, during lactation until after weaning and compared with control fed counterparts. Liver specimens were analyzed for regulated proteins using 2‐DE and MALDI‐TOF‐MS and plasma samples for metabolites. Based on the 26 differentially expressed proteins associated with depleted liver glycogen content, elevated urea and citrulline plasma concentrations, we conclude that HP feeding during lactation leads to an activated amino acid, carbohydrate and fatty acid catabolism while it activates gluconeogenesis. From pregnancy to lactation, plasma arginine, tryptophan, serine, glutamine and cysteine decreased, whereas urea concentrations increased in both groups. Concomitantly, hepatic glycogen content decreased while total fat content remained unaltered in both groups. Consideration of 59 proteins differentially expressed between pregnancy and lactation highlights different strategies of HP and control fed mice to meet energy requirements for lactation by adjusting amino acid degradation, carbohydrate and fat metabolism, citrate cycle, but also ATP‐turnover, protein folding, secretion of proteins and (de)activation of transcription factors.

IGFBP-2 overexpression reduces the appearance of dysplastic aberrant crypt foci and inhibits growth of adenomas in chemically induced colorectal carcinogenesis.

Colon cancer patients frequently show increased levels of serum insulin‐like growth factor‐binding protein‐2 (IGFBP‐2), however, the pathogenetic relevance of this phenomenon for colorectal cancer is unclear. Therefore, we have used IGFBP‐2 transgenic animals which overexpress IGFBP‐2 systemically and locally in the intestine to study its role in chemically induced colorectal carcinogenesis. Mice received intraperitoneal injections of 1,2‐dimethylhydrazine (DMH) (40 mg/kg body weight) once a week for 6 weeks to selectively induce aberrant crypt foci (ACF) and tumors in the colon. While tumor incidence was comparable in transgenic and control mice, the volume of adenomas in IGFBP‐2 transgenic mice was reduced more than 2‐fold. Furthermore, serum IGFBP‐2 levels negatively correlated with tumor volume in the IGFBP‐2 transgenic group. Histological examination showed that IGFBP‐2 transgenic mice developed significantly less dysplastic ACF with a high potential to progress to advanced stages. The reduced tumor volume in IGFBP‐2 transgenic animals was due to significantly reduced proliferative capacity, evidenced by a lower proportion of cells positive for Ki67. Our results demonstrate for the first time in an experimental model that IGFBP‐2 overabundance prior to the onset and during colorectal carcinogenesis reduces tumor growth by inhibition of cell proliferation.

Genetic-statistical analysis of growth in selected and unselected mouse lines

Summary The growth of males sampled from two mouse lines long-term selected for over 86 generations on body weight (DU6) or on protein amount (DU6P) was analysed from birth till 120 days of age and compared to the growth of an unselected control line (DUKs). Animals from the selected lines are already approximately 40 to 50% heavier at birth than the controls. This divergence increases to about 210 to 240% at the 120 day of age. With birth weights of 2.2 and 2.4 g and weights of 78 and 89 g at the 120 day these selection lines are the heaviest known mouse lines. The fit of three modified non-linear growth functions ( Gompertz function, Logistic function, Richards function) was compared and the effect of three different data inputs elucidated. The modification was undertaken to use parameters having a direct biological meaning, for example: A: theoretical final body weight, B: maximum weight gain, C: age at maximum weight gain, D (only Richards function): determines the position of the inflection point in relation to the final weight. All three models fit the observed data very well (r 2 = 0.949–0.998), with a slight advantage for the Richards function. There were no substantial effects of the data input (averages, single values, fitting a curve for every animal with subsequent averaging the parameters). The high growth of the selected mice is connected with very substantial changes in the final weight and in the maximum weight gain, whereas the changes of the age at the point of inflection were, although partially significant, relatively small and dependent on the model used.

Influence of chlorocholinechloride-treated wheat on selected in vitro fertility parameters in male mice.

The aim of this study was to evaluate the influence of feeding with food and water containing chlorocholinechloride (CCC) on the fertility of male mice in a two-generation study. For this purpose the number of testicular spermatozoa and the relative proportion of primary and secondary spermatocytes involved in spermatogenesis were measured. Furthermore, the fertility of epididymal spermatozoa from tested male mice was investigated in a special in-vitro fertilization system. The experimental food was composed of CCC-treated wheat in the first experiment and CCC-free wheat and water mixed with pure CCC in the second experiment. The CCC residue content in the treated food and water was 0.21 mg/kg and 0.2 mg/L, respectively. Under the influence of feeding with CCC-treated wheat (Experiment 1) the fertilization and cleavage rates of oocytes incubated with spermatozoa from CCC-fed mice were reduced: the fertilization rate 65.1% vs. 21.1% and the cleavage rate 51.9% vs. 20.3%, p < 0.01 (control feeding vs. CCC feeding, respectively). Feeding of sperm donors with pure CCC mixed with untreated wheat pellets or water (Experiment 2) led to a reduction in the fertilization and cleavage rate (control: 60.8%, 32.4%; CCC-food: 29.8%, 12.1%; CCC-water: 30.1%, 10.2%; CCC-food/water: 36.6%, 12.5%; p < 0.01, respectively). The normal course of spermatogenesis was unchanged after the exposure to CCC. Testicular weight, the number of spermatozoa, and the proportion of haploid, diploid, and tetraploid testicular cells were not influenced. However, the functional competence of epididymal spermatozoa from CCC-fed donors was reduced, resulting in a significantly diminished fertilization and cleavage rate in vitro. The results suggest that CCC could interfere with epididymal protein secretion and the process of sperm maturation during passage through the epididymis.

Metabolic responses to high-fat diets rich in n-3 or n-6 long-chain polyunsaturated fatty acids in mice selected for either high body weight or leanness explain different health outcomes

BackgroundIncreasing evidence suggests that diets high in polyunsaturated fatty acids (PUFA) confer health benefits by improving insulin sensitivity and lipid metabolism in liver, muscle and adipose tissue.MethodsThe present study investigates metabolic responses in two different lines of mice either selected for high body weight (DU6) leading to rapid obesity development, or selected for high treadmill performance (DUhTP) leading to a lean phenotype. At 29 days of age the mice were fed standard chow (7.2% fat, 25.7% protein), or a high-fat diet rich in n-3 PUFA (n-3 HFD, 27.7% fat, 19% protein) or a high-fat diet rich in n-6 PUFA (n-6 HFD, 27.7% fat, 18.6% protein) for 8 weeks. The aim of the study was to determine the effect of these PUFA-rich high-fat diets on the fatty acid profile and on the protein expression of key components of insulin signalling pathways.ResultsPlasma concentrations of leptin and insulin were higher in DU6 in comparison with DUhTP mice. The high-fat diets stimulated a strong increase in leptin levels and body fat only in DU6 mice. Muscle and liver fatty acid composition were clearly changed by dietary lipid composition. In both lines of mice n-3 HFD feeding significantly reduced the hepatic insulin receptor β protein concentration which may explain decreased insulin action in liver. In contrast, protein kinase C ζ expression increased strongly in abdominal fat of n-3 HFD fed DUhTP mice, indicating enhanced insulin sensitivity in adipose tissue.ConclusionsA diet high in n-3 PUFA may facilitate a shift from fuel deposition in liver to fuel storage as fat in adipose tissue in mice. Tissue specific changes in insulin sensitivity may describe, at least in part, the health improving properties of dietary n-3 PUFA. However, important genotype-diet interactions may explain why such diets have little effect in some population groups.

Two high-fertility mouse lines show differences in component fertility traits after long-term selection

UNLABELLED Two selected high-fertility mouse lines, namely FL1 and FL2, and a non-selected control (Fzt:DU), all derived from the same genetic pool, were analysed as an animal model for polytocous species to elucidate the effects of long-term selection and to identify relevant component traits that may be responsible for fertility performance. The index trait used for breeding selection was largely increased by 104% and 142% in the FL1 and FL2 lines, respectively, resulting in an average litter size of 17.3 pups and 18.7 pups per litter in the FL1 and FL2 lines, respectively, compared with a litter size of 11.0 pups per litter in the control (Fzt:DU). In addition, different component fertility traits were analysed in females of all three lines at different stages of the oestrous cycle and pregnancy. IN CONCLUSION (1) early embryonic development was accelerated in the FL1 and FL2 lines compared with control; (2) plasma progesterone levels were not correlated with fertility performance; (3) a largely increased ovulation number (i.e. number of corpora lutea) was responsible for high prolificacy in both lines; however, (4) the number of ova shed, as well as the rate of loss of ova and pre- and postimplantation conceptuses, was very different in the FL1 and FL2 lines, suggesting that different genetic components may be responsible for the high prolificacy in both high-fertility lines.

Expression profiling of a high-fertility mouse line by microarray analysis and qPCR

BackgroundIn a recent study it was demonstrated that a largely increased ovulation number is responsible for high prolificacy in two mouse lines selected for fertility performance. The objective of the present study was to identify genes that are involved in increasing the ovulation number in one of these lines, FL1. For differential expression profiling, ovaries of FL1 and of a non-selected control line, DUKsi, both lines derived from the same genetic pool, were analyzed with microarray analysis and quantitative polymerase chain reaction (qPCR). Ovaries from 30 animals of each line were collected at the metestrous stage, combined to 6 pools each, and processed for microarray analysis.ResultsThe actual number of ova shed in FL1 exceeded that of the DUKsi control line more than twofold (26.6 vs. 12.9). 148 differentially expressed ovarian transcripts could be identified, 74 of them up- and 74 down-regulated. Of these, 47 significantly mapped to specific Gene Ontology (GO) terms representing different biological processes as steroid metabolism, folliculogenesis, immune response, intracellular signal transduction (particularly of the G protein signaling cascade), regulation of transcription and translation, cell cycle and others. qPCR was used to re-evaluate selected transcripts and to estimate inter-individual variation of expression levels. These data significantly correlated with microarray data in 12 out of 15 selected transcripts but revealed partly large variations of expression levels between individuals.Conclusion(1) The abundance of numerous ovarian transcripts was significantly different in FL1 compared to the non-selected control line DUKsi thus suggesting that at least some of the respective genes and corresponding biological processes are involved in improving reproductive traits, particularly by increasing the number of ovulation. (2) Selective qPCR re-evaluation largely confirmed the microarray data and in addition demonstrated that sample pooling can be beneficial to find out group-specific expression profiles despite of large inter-individual variation. (3) The present data will substantially help ongoing genetic association studies to identify candidate genes and causative mutations responsible for increased fertility performance in mice.

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.

High-fat diets rich in ω-3 or ω-6 polyunsaturated fatty acids have distinct effects on lipid profiles and lipid peroxidation in mice selected for either high body weight or leanness

OBJECTIVE The aim of the study is to determine the response of muscle lipid peroxidation and the fatty-acid profile of three groups of mice-high body weight (DU6) obesity-prone mice, high treadmill performance (DUhTP) lean mice, and unselected control mice (DUK) fed high-fat diets (HFDs) rich in ω-3 or ω-6 polyunsaturated fatty acids (PUFA). METHODS The isocaloric HFDs were enriched with either ω-3 PUFA (27% fish oil, ω-3 HFD) or ω-6 PUFA (27% sunflower oil, ω-6 HFD), and the control group was fed standard chow (7.2% fat). Statistical calculations were done with procedure GLM of SAS. RESULTS As expected, the ω-3 and ω-6 PUFA-rich HFDs showed significant effects on fatty-acid concentrations of skeletal muscle in all three lines of mice compared with the standard chow. The investigations of muscle lipid peroxidation revealed that the ω-3 PUFA-rich HFD caused the highest lipid peroxidation values in muscle of lean DUhTP mice and unselected control DUK mice. However, lower lipid peroxidation levels were observed in the obesity-prone DU6 mice. In contrast, the ω-6 PUFA-rich HFD did not influence lipid peroxidation in muscle of any of the different lines of mice. The present study suggests that a higher overall antioxidant capacity in the muscle tissue of obesity-prone DU6 mice may lead to lower levels of reactive oxygen species formation by ω-3 PUFA-rich HFDs in comparison with lean DUhTP mice. CONCLUSION These studies raise the possibility that obesity per se may be protective against oxidative damage when high ω-3 PUFA diets are used.