Quantitative trait loci for carbohydrate and total energy intake on mouse chromosome 17: congenic strain confirmation and candidate gene analyses (Glo1, Glp1r)

Quantitative trait loci (QTL) for carbohydrate (Mnic1) and total energy (Kcal2) intake on proximal mouse chromosome 17 were identified previously from a C57BL/6J (B6) X CAST/Ei (CAST) intercross. Here we report that a new congenic strain developed in our laboratory has confirmed this complex locus by recapitulating the original linked phenotypes: B6.CAST-17 homozygous congenic mice consumed more carbohydrate (27%) and total energy (17%) compared with littermate wild-type mice. Positional gene candidates with relevance to carbohydrate metabolism, glyoxalase I (Glo1) and glucagon-like peptide-1 receptor (Glp1r), were evaluated. Glo1 expression was upregulated in liver and hypothalamus of congenic mice when compared with B6 mice. Analyses of Glp1r mRNA and protein expression revealed tissue-specific strain differences in pancreas (congenic>B6) and stomach (B6>congenic). These results suggest the possibility of separate mechanisms for enhanced insulin synthesis and gastric accommodation in the presence of high carbohydrate intake and larger food volume, respectively. Sequence analysis of Glp1r found a G insert at nt position 1349, which results in earlier termination of the open reading frame, thus revealing an error in the public sequence. Consequently, the predicted length of GLP-1R is 463 aa compared with 489 aa, as previously reported. Also, we found a polymorphism in Glp1r between parental strains that alters the amino acid sequence. Variation in Glp1r could influence nutrient intake in this model through changes in the regulatory or protein coding regions of the gene. These congenic mice offer a powerful tool for investigating gene interactions in the control of food intake.     

Increased proliferative activity in selenium-deficient mouse liver

Male albino NMRI mice were fed a selenium-deficient (Se-), torula yeast-based diet containing less than 10 ppb Se for at least 2 months (Se-) while a control group received the same diet supplemented (Se+) with 330 ppb Se as Na2SeO3. The Se-(-)animals showed multiple enzyme modulations of liver enzyme activities indicating that they were in a severely Se- state. No significant difference in the basic DNA synthesis rate of Se-(-)animals compared to Se+ controls was measured. However, when liver cell proliferation was induced by either hepatopoietin pretreatment or by partial hepatectomy, an about 3-fold increase in DNA replication rates was found in Se- compared to controls. We conclude that the enhanced proliferative activity in Se- mouse liver is expressed in an emergency situation.     

Increased daily physical activity and fatigue symptoms in chronic fatigue syndrome

Individuals with chronic fatigue syndrome (CFS) have been shown to have reduced activity levels associated with heightened feelings of fatigue. Previous research has demonstrated that exercise training has beneficial effects on fatigue-related symptoms in individuals with CFS. PURPOSE: The aim of this study was to sustain an increase in daily physical activity in CFS patients for 4 weeks and assess the effects on fatigue, muscle pain and overall mood. METHODS: Six CFS and seven sedentary controls were studied. Daily activity was assessed by a CSA accelerometer. Following a two week baseline period, CFS subjects were asked to increase their daily physical activity by 30% over baseline by walking a prescribed amount each day for a period of four weeks. Fatigue, muscle pain and overall mood were reported daily using a 0 to 100 visual analog scale and weekly using the Profile of Mood States (Bipolar) questionnaire. RESULTS: CFS patients had significantly lower daily activity counts than controls (162.5 +/- 51.7 x 103 counts/day vs. 267.2 +/- 79.5 x 103 counts/day) during a 2-week baseline period. At baseline, the CFS patients reported significantly (P < 0.01) higher fatigue and muscle pain intensity compared to controls but the groups did not differ in overall mood. CFS subjects increased their daily activity by 28 +/- 19.7% over a 4 week period. Overall mood and muscle pain worsened in the CFS patients with increased activity. CONCLUSION: CFS patients were able to increase their daily physical activity for a period of four weeks. In contrast to previous studies fatigue, muscle pain, and overall mood did not improve with increased activity. Increased activity was not presented as a treatment which may account for the differential findings between this and previous studies. The results suggest that a daily "activity limit" may exist in this population. Future studies on the impact of physical activity on the symptoms of CFS patients are needed.     

Dissection of a genetically complex cluster of growth and obesity QTLs on mouse chromosome 2 using subcongenic intercrosses

In a previous study we characterized the B6.CAST-(D2Mit329-D2Mit457)N(6) (B62D) congenic strain, which possesses CAST/EiJ (CAST) chromosome 2 donor alleles from 74 to 180 Mbp on a C57BL6/J (B6) background. This strain exhibited significant decreases in body weight and adiposity attributable to the weight gain 2 (Wg2) quantitative trait locus (QTL). To refine the location of Wg2, we used a two-stage genetic dissection strategy consisting of a B62D × B6 backcross, which mapped Wg2 to the proximal portion of the B62D donor region, followed by the development of seven overlapping subcongenic F₂ intercrosses targeting the Wg2 genomic interval. Surprisingly, five of the seven intercrosses displayed significant differences, dependent on genotype, in body weight and/or fat pad mass. These effects were the result of at least four independent QTLs that were named Wg2a, b, c, and d. In contrast to the lean and low body weight phenotype of the B62D parental strain, mice homozygous for CAST congenic alleles (cast/cast) at Wg2a were significantly heavier at 6 and 9 weeks of age, while cast/cast mice at Wg2c had higher levels of total fat. Consistent with the prior observed effects of Wg2, cast/cast mice at Wg2b displayed significant decreases in 6- and 9-week body weight as well as a decrease in total fat pad mass. All of the QTLs had additive effects on body composition except Wg2d, which displayed underdominance for total fat mass. Significant differences in weight and adiposity were also observed in genetically identical b6/b6 homozygous mice across the panel of subcongenics, suggesting either maternal or paternal contributions to body composition. These data represent a significant advancement toward the identification of mouse chromosome 2 growth and obesity quantitative trait genes.     

Increased genetic variance of BMI with a higher prevalence of obesity

Background and objectives

There is no doubt that the dramatic worldwide increase in obesity prevalence is due to changes in environmental factors. However, twin studies suggest that genetic differences are responsible for the major part of the variation in body mass index (BMI) and other measures of body fatness within populations. Several recent studies suggest that the genetic effects on adiposity may be stronger when combined with presumed risk factors for obesity. We tested the hypothesis that a higher prevalence of obesity and overweight and a higher BMI mean is associated with a larger genetic variation in BMI.

Methods

The data consisted of self-reported height and weight from two Danish twin surveys in 1994 and 2002. A total of 15,017 monozygotic and dizygotic twin pairs were divided into subgroups by year of birth (from 1931 through 1982) and sex. The genetic and environmental variance components of BMI were calculated for each subgroup using the classical twin design. Likewise, the prevalence of obesity, prevalence of overweight and the mean of the BMI distribution was calculated for each subgroup and tested as explanatory variables in a random effects meta-regression model with the square root of the additive genetic variance (equal to the standard deviation) as the dependent variable.

Results

The size of additive genetic variation was positively and significantly associated with obesity prevalence (p = 0.001) and the mean of the BMI distribution (p = 0.015). The association with prevalence of overweight was positive but not statistically significant (p = 0.177).

Conclusion

The results suggest that the genetic variation in BMI increases as the prevalence of obesity, prevalence of overweight and the BMI mean increases. The findings suggest that the genes related to body fatness are expressed more aggressively under the influence of an obesity-promoting environment.     

Rhodanese activity and total sulfur content in frog and mouse liver

The activity of rhodanese was histochemically tested in cryostat sections of the frog (Rana temporaria) and mouse liver. The activity levels were evaluated in sections, and the results were expressed as the ratio of the area of all granules, products of the enzymatic test, to the total analyzed area (area fraction). The present study confirmed the biochemically detected activity of rhodanese, and showed a large pool of endogenous sulfane sulfur donors, substrates for rhodanese, in the frog liver. The area of a single granule corresponded to the size of the mitochondrium, what suggests enzyme localization in this organelle. In view of this it should be considered whether the rhodanese activity, biochemically detected in the cytosolic fraction of the frog liver, results from the enzyme action. The total content of sulfur in cryostat sections of the mouse and frog liver was calculated and compared on the basis of the Energy Dispersion Spectrum (EDS) obtained by a scanning microscope. These studies showed higher total sulfur content in the frog liver than in the mouse liver. The high total content of sulfur in the frog liver in autumn might be associated with sulfur storing for protein biosynthesis during the period of hibernation.     

Exhaustive physical exercise and acid hydrolase activity in mouse skeletal muscle

Summary Adult, untrained NMRI mice were exhausted on a motor-driven treadmill by an intermittent-type running programme. Serial cryostate sections for the staining of NADH-tetrazolium reductase, -glucuronidase, -N-acetylglucosaminidase, and -glycerophosphatase activities and for making hematoxylin-eosin staining were cut from m. quadriceps femoris 1, 2, 3, 5, 7, and 15 days after physical exhaustion. A strong increase in the activities of -glucuronidase and -N-acetylglucosaminidase, was observed 7 days after exhaustion and the activity changes, which were similar for the both glycosidases, were more prominent in the highly oxidative red compared to less oxidative white fibres. Activity granules were more numerous in the perinuclear than the interfibrillar area of red fibres. Spots were arranged like longitudinal chains between myofibrils. Activity in connective tissue was usually observed only in animals exhausted 3–7 days earlier. Simultaneous activity in fibres exceeded that in connective tissue -Glycerophosphatase activity was not, by the method used, seen in histologically healthy or normal-looking fibres. in samples taken 2–5 days after exhaustion some degenerating and necrotic fibres were observed. Inflammatory reaction was also observed being at its strongest five days after loading when mononuclear cells were seen inside necrotic fibres. The number of regenerating muscle cells was most abundant 7 days after exhaustion. It is suggested that temporary hypoxia, which accompanies exhaustive physical exercise in skeletal muscle, upsets the energy metabolism and homeostasis of fibres and causes the observed histological and histochemical alterations, which posses features typical of both lethal and sublethal acute cell injury.     

Increased 5-enolpyruvylshikimic acid 3-phosphate synthase activity in a glyphosate-tolerant variant strain of tomato cells

A glyphosate-tolerant variant of cultured tomato cells (Lycopersicon esculentum × L. peruvianum hybrid) was isolated via a single-step selection. Growth of the variant in suspension culture was essentially unaffected by 10 mM glyphosate, 100 times the concentration needed to significantly reduce the growth rate of wild type cells. When treated with glyphosate, variant cells accumulated much less shikimic acid than did the wild type cells. In analyses of 5-enolpyruvyl-shikimic acid 3-phosphate (EPSP) synthase activity in two separate experiments, the variant cells had 8 and 13 times higher specific activity than the wild type cells. The enzyme activities from the two types of cells were equally inhibited by glyphosate. These results suggest that the glyphosate tolerance of the variant results from overaccumulation of a glyphosate-sensitive EPSP synthase. Attempts to regenerate fertile plants from the variant cells were unsuccessful, but abnormal shoots were regenerated and callus from leaves of these shoots retained the tolerance to glyphosate.     

An estradiol-responsive mouse endometrial cell strain with inducible aryl hydrocarbon hydroxylase activity

A mouse endometrial cell population has been isolated by mild tryptic digestion of the uterine lining. The cells were morphologically similar to endometrial gland cells in the intact mouse endometrial gland. The endometrial cells had a modal chromosome number of 66. The cells were adherent to glass as well as plastic and contained numerous large refractile, osmophilic, non-membrane-limited granules which stain with periodic acid-Schiff reagent but do not stain with oil red O, Sudan black, or Alcian blue. Cell growth was responsive to 17β-estradiol; cell number increased 1.34-fold in 4 days in the presence of 10^?8 M estradiol. The cells are not tumorigenic. The cells showed induction of aryl hydrocarbon hydroxylase (AHH) activity when 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was added to the growth media for 24 h. AHH activity and its induction were investigated with cells grown in the presence and absence 10^?8 M estradiol. Cells grown in media containing estradiol exhibited a 6.2-fold induction by TCDD; cells grown without estradiol gave an 8.4-fold induction of AHH activity. AHH activity and its induction by TCDD were demonstrated in cells grown with fetal calf serum that had been pretreated with dextran-coated charcoal to remove endogenous steroids. Benzanthracene failed to induce AHH activity significantly.     

A Myo7a mutation cosegregates with stereocilia defects and low-frequency hearing impairment

A phenotype-driven approach was adopted in the mouse to identify molecules involved in ear development and function. Mutant mice were obtained using N-ethyl-N-nitrosourea (ENU) mutagenesis and were screened for dominant mutations that affect hearing and/or balance. Heterozygote headbanger (Hdb/+) mutants display classic behavior indicative of vestibular dysfunction including hyperactivity and head bobbing, and they show a Preyer reflex in response to sound but have raised cochlear thresholds especially at low frequencies. Scanning electron microscopy of the surface of the organ of Corti revealed abnormal stereocilia bundle development from an early age that was more severe in the apex than the base. Utricular stereocilia were long, thin, and wispy. Homozygotes showed a similar but more severe phenotype. The headbanger mutation has been mapped to a 1.5-cM region on mouse Chromosome 7 in the region of the unconventional myosin gene Myo7a, and mutation screening revealed an A>T transversion that is predicted to cause an isoleucine-to-phenylalanine amino acid substitution (I178F) in a conserved region in the motor-encoding domain of the gene. Protein analysis revealed reduced levels of myosin VIIa expression in inner ears of headbanger mice. Headbanger represents a novel inner ear phenotype and provides a potential model for low-frequency-type human hearing loss.These authors contributed equally to this study.     

Investigation of circadian rhythms in a genetically anophthalmic mouse strain: Correlation of activity patterns with suprachiasmatic nuclei hypogenesis

Summary ZRDCT-An mice are anophthalmic mutants with varying degrees of hypogenesis of the mediobasal hypothalamus and suprachias-matic nuclei. Statistical analysis of the wheelrunning activity patterns of these animals showed that most mice had circadian activity rhythms unentrained to the light-dark cycle. However, four of 22 animals had random or non-circadian activity patterns. Of these four mice, three had fewer than one third the typical number of cells in the suprachiasmatic nuclei. The absence of detectable circadian rhythms in mice with genetically produced hypogenesis of the suprachiasmatic nuclei is reminiscent of the effects of partial electrolytic lesions of the same tissue.Abbreviations SCN suprachiasmatic nuclei - LD light dark - DD dark-dark     

Increased physical activity severely induces osteoarthritic changes in knee joints with papain induced sulfate-glycosaminoglycan depleted cartilage

Introduction

Articular cartilage needs sulfated-glycosaminoglycans (sGAGs) to withstand high pressures while mechanically loaded. Chondrocyte sGAG synthesis is regulated by exposure to compressive forces. Moderate physical exercise is known to improve cartilage sGAG content and might protect against osteoarthritis (OA). This study investigated whether rat knee joints with sGAG depleted articular cartilage through papain injections might benefit from moderate exercise, or whether this increases the susceptibility for cartilage degeneration.

Methods

sGAGs were depleted from cartilage through intraarticular papain injections in the left knee joints of 40 Wistar rats; their contralateral joints served as healthy controls. Of the 40 rats included in the study, 20 rats remained sedentary, and the other 20 were subjected to a moderately intense running protocol. Animals were longitudinally monitored for 12 weeks with in vivo micro-computed tomography (μCT) to measure subchondral bone changes and single-photon emission computed tomography (SPECT)/CT to determine synovial macrophage activation. Articular cartilage was analyzed at 6 and 12 weeks with ex vivo contrast-enhanced μCT and histology to measure sGAG content and cartilage thickness.

Results

All outcome measures were unaffected by moderate exercise in healthy control joints of running animals compared with healthy control joints of sedentary animals. Papain injections in sedentary animals resulted in severe sGAG-depleted cartilage, slight loss of subchondral cortical bone, increased macrophage activation, and osteophyte formation. In running animals, papain-induced sGAG-depleted cartilage showed increased cartilage matrix degradation, sclerotic bone formation, increased macrophage activation, and more osteophyte formation.

Conclusions

Moderate exercise enhanced OA progression in papain-injected joints and did not protect against development of the disease. This was not restricted to more-extensive cartilage damage, but also resulted in pronounced subchondral sclerosis, synovial macrophage activation, and osteophyte formation.     

Effect of exercise training on total daily physical activity in elderly humans

This study examined the effect of 12 weeks of exercise training on daily physical activity in elderly humans. Training consisted of a weekly group session and an individual session with cardio- and weight-stack machines. A group of 15 subjects served as the exercise group [EXER mean age 59 (SD 4) years], and 7 subjects as the controls [CONT mean age 57 (SD 3) years]. Physical activity and physical fitness were measured before the start of training (T), at week 6 and week 12 (T0, T6, T12 respectively) in EXER, and at T0 and T12 in CONT. Physical activity over 14 days was measured using a tri-axial accelerometer and physical fitness was measured during an incremental exercise test. At T12, mean maximal power output had significantly increased in EXER compared to CONT 8 (SD 12) vs -5 (SD 9) W; P < 0.02] and mean submaximal heart rate (at 100 W) had reduced [-10 (SD 7) vs -2 (SD 6) beats x min(-1); P < 0.05]. No differences or changes in physical activity were observed between EXER and CONT. At T6, physical activity on training days was significantly higher than on non-training days (P < 0.001). When the accelerometer output of the training session was subtracted from the accelerometer output on training days, at T12 non-training physical activity was significantly lower than on non-training days (P < 0.004). Accelerometer output of the individual training session at T12 had significantly increased compared to T6 (P < 0.05), whereas, accelerometer output of the group training session had remained unchanged. In conclusion, in elderly subjects an exercise training programme of moderate intensity resulted in an improved physical fitness but had no effect on total daily physical activity. Training activity was compensated for by a decrease in non-training physical activity.