Emily E. Schmitt

Vivo-Morpholinos Induced Transient Knockdown of Physical Activity Related Proteins

Physical activity is associated with disease prevention and overall wellbeing. Additionally there has been evidence that physical activity level is a result of genetic influence. However, there has not been a reliable method to silence candidate genes in vivo to determine causal mechanisms of physical activity regulation. Vivo-morpholinos are a potential method to transiently silence specific genes. Thus, the aim of this study was to validate the use of Vivo-morpholinos in a mouse model for voluntary physical activity with several sub-objectives. We observed that Vivo-morpholinos achieved between 60–97% knockdown of Drd1-, Vmat2-, and Glut4-protein in skeletal muscle, the delivery moiety of Vivo-morpholinos (scramble) did not influence physical activity and that a cocktail of multiple Vivo-morpholinos can be given in a single treatment to achieve protein knockdown of two different targeted proteins in skeletal muscle simultaneously. Knocking down Drd1, Vmat2, or Glut4 protein in skeletal muscle did not affect physical activity. Vivo-morpholinos injected intravenously alone did not significantly knockdown Vmat2-protein expression in the brain (p = 0.28). However, the use of a bradykinin analog to increase blood-brain-barrier permeability in conjunction with the Vivo-morpholinos significantly (p = 0.0001) decreased Vmat2-protein in the brain with a corresponding later over-expression of Vmat2 coincident with a significant (p = 0.0016) increase in physical activity. We conclude that Vivo-morpholinos can be a valuable tool in determining causal gene-phenotype relationships in whole animal models.

Differential skeletal muscle proteome of high- and low-active mice.

Physical inactivity contributes to cardiovascular disease, type II diabetes, obesity, and some types of cancer. While the literature is clear that there is genetic regulation of physical activity with existing gene knockout data suggesting that skeletal muscle mechanisms contribute to the regulation of activity, actual differences in end-protein expression between high- and low-active mice have not been investigated. This study used two-dimensional differential gel electrophoresis coupled with mass spectrometry to evaluate the proteomic differences between high-active (C57L/J) and low-active (C3H/HeJ) mice in the soleus and extensor digitorum longus (EDL). Furthermore, vivo-morpholinos were used to transiently knockdown candidate proteins to confirm their involvement in physical activity regulation. Proteins with higher expression patterns generally fell into the calcium-regulating and Krebs (TCA) cycle pathways in the high-active mice (e.g., annexin A6, P = 0.0031; calsequestrin 1; P = 0.000025), while the overexpressed proteins in the low-active mice generally fell into cytoskeletal structure- and electron transport chain-related pathways (e.g., ATPase, P = 0.031; NADH dehydrogenase, P = 0.027). Transient knockdown of annexin A6 and calsequestrin 1 protein of high-active mice with vivo-morpholinos resulted in decreased physical activity levels (P = 0.001). These data suggest that high- and low-active mice have unique protein expression patterns and that each pattern contributes to the peripheral capability to be either high- or low-active, suggesting that different specific mechanisms regulate activity leading to the high- or low-activity status of the animal.

Nck deficiency is associated with delayed breast carcinoma progression and reduced metastasis

Nck promotes breast carcinoma progression and metastasis by directing the polarized interaction of carcinoma cells with collagen fibrils, decreasing actin turnover, and enhancing the localization and activity of MMP14 at the cell surface through modulation of the spatiotemporal activation of Cdc42 and RhoA.

Differential protein expression in the nucleus accumbens of high and low active mice.

Physical inactivity is associated with the development of a variety of chronic illnesses. Literature has shown that physical activity is genetically regulated; however there is limited information on the mechanisms that influence this process with existing studies primarily focused on genomic and/or transcription association studies. There have been no studies to determine differential protein expression in the nucleus accumbens, the brain site thought to be involved in activity regulation, between high and low active animals. We compared the global nucleus accumbens proteome signature from known high- and low-active mice and identified seven differentially expressed proteins. Low active mice generally over expressed proteins associated with neural stress (Stress 70 protein and V type proton ATPase catalytic subunit A), and the high-active mice over expressed proteins associated with metabolism (creatine kinase B, succinyl-CoA ligase). Previously suggested mechanisms associated with activity regulation in the nucleus accumbens have centered on dopamine receptor 1 and endocannabinoid receptor 1. However, these proteins and the associated pathways were not differentially expressed between high and low active mice. In conclusion, protein expression must be determined as part of the effort to identify involved mechanisms in regulating activity and there appears to be separate nucleus accumbens proteome signatures associated with high- and low-active mice.

Circadian Regulation of Benzo[a]Pyrene Metabolism and DNA Adduct Formation in Breast Cells and the Mouse Mammary Gland

The circadian clock plays a role in many biologic processes, yet very little is known about its role in metabolism of drugs and carcinogens. The purpose of this study was to define the impact of circadian rhythms on benzo-a-pyrene (BaP) metabolism in the mouse mammary gland and develop a circadian in vitro model for investigating changes in BaP metabolism resulting from cross-talk between the molecular clock and aryl hydrocarbon receptor. Female 129sv mice (12 weeks old) received a single gavage dose of 50 mg/kg BaP at either noon or midnight, and mammary tissues were isolated 4 or 24 hours later. BaP-induced Cyp1a1 and Cyp1b1 mRNA levels were higher 4 hours after dosing at noon than at 4 hours after dosing at midnight, and this corresponded with parallel changes in Per gene expression. In our in vitro model, we dosed MCF10A mammary cells at different times after serum shock to study how time of day shifts drug metabolism in cells. Analysis of CYP1A1 and CYP1B1 gene expression showed the maximum enzyme-induced metabolism response 12 and 20 hours after shock, as determined by ethoxyresorufin-O-deethylase activity, metabolism of BaP, and formation of DNA-BaP adducts. The pattern of PER-, BMAL-, and aryl hydrocarbon receptor–induced P450 gene expression and BaP metabolism was similar to BaP-induced Cyp1A1 and Cyp1B1 and molecular clock gene expression in mouse mammary glands. These studies indicate time-of-day exposure influences BaP metabolism in mouse mammary glands and describe an in vitro model that can be used to investigate the circadian influence on the metabolism of carcinogens.

Environmental Endocrine Disruptor Affects Voluntary Physical Activity in Mice.

INTRODUCTION Voluntary physical activity levels are regulated by sex hormones. The purpose of this study was to determine the effect of the endocrine disruptor benzyl butyl phthalate (BBP) on the regulation of physical activity in mice. METHODS Mouse dams were treated with 500 mg·kg·d of BBP or vehicle on gestation days 9-16. Pups were weaned and analyzed for voluntary physical activity levels, puberty development, sex hormone levels, and body composition during the 20-wk period. RESULTS Seventy-three offspring from BBP-treated dams were studied (n = 43 males and n = 30 females). Endocrine disruption was indicated by decreased anogenital distances in BBP-treated male offspring at 10 (P = 0.001) and 20 wk (P = 0.038) and delayed vaginal openings in BBP-treated female offspring (P = 0.001). Further, there was a significant decrease in serum testosterone concentration in male mice between control and BBP at 10 wk (P = 0.039) and at 20 wk (P = 0.022). In female mice, there was a significant increase in serum testosterone concentration in BBP mice at 20 wk (P = 0.002) and a significant increase in estrogen (estradiol) concentrations at 20 wk in the control female mice (P = 0.015). Overall, BBP mice ran significantly less distance (males, P = 0.008; females, P = 0.042) than controls. Other than a significant increase in BBP-treated males in fat mass at 20 wk (P = 0.040), there was no significant decrease in weight, lean mass, or fat mass in either female or male mice, regardless of treatment. CONCLUSION Maternal endocrine disruption altered hormone response, but not body composition in either sex of offspring, with a corresponding decreased activity throughout early adulthood in all offspring. These results suggest that exposure to common environmental endocrine disruptors in utero can reduce and alter physical activity levels in offspring.

PER2 regulation of mammary gland development

The molecular clock plays key roles in daily physiological functions, development and cancer. Period 2 (PER2) is a repressive element, which inhibits transcription activated by positive clock elements, resulting in diurnal cycling of genes. However, there are gaps in our understanding of the role of the clock in normal development outside of its time-keeping function. Here, we show that PER2 has a noncircadian function that is crucial to mammalian mammary gland development. Virgin Per2-deficient mice, Per2−/−, have underdeveloped glands, containing fewer bifurcations and terminal ducts than glands of wild-type mice. Using a transplantation model, we show that these changes are intrinsic to the gland and further identify changes in cell fate commitment. Per2−/− mouse mammary glands have a dual luminal/basal phenotypic character in cells of the ductal epithelium. We identified colocalization of E-cadherin and keratin 14 in luminal cells. Similar results were demonstrated using MCF10A and shPER2 MCF10A human cell lines. Collectively this study reveals a crucial noncircadian function of PER2 in mammalian mammary gland development, validates the Per2−/− model, and describes a potential role for PER2 in breast cancer. Summary: The molecular clock is associated with crucial biological processes, but little is known about its role in development. Here, we show that PER2 plays a key role in mammary gland development.

Weekly Physical Activity Levels of Older Adults Regularly Using a Fitness Facility.

The aim of this paper was to determine if weekly physical activity levels were greater in an independent-living older adult population that was regularly participating in structured fitness activities. Also, lifetime exercise history and sex differences were investigated in an effort to understand how they relate to current weekly step activity. Total weekly step counts, measured with a pedometer, were assessed in two older adult groups; the first consisted of members of a local senior center who regularly used the fitness facility (74.5 ± 6.0 yrs; mean ± SD) while the second group consisted of members who did not use the fitness facility (74.8 ± 6.0 yrs). Participants also completed the Lifetime Physical Activity Questionnaire (LPAQ). No significant difference was found in the total number of weekly steps between groups (p = 0.88) or sexes (p = 0.27). The LPAQ suggested a significant decline in activity with aging (p = 0.01) but no difference between groups (p = 0.54) or sexes (p = 0.80). A relationship was observed between current step activity and MET expenditure over the past year (p = 0.008, r 2 = 0.153) and from ages 35 to 50 years (p = 0.037, r 2 = 0.097). The lack of difference in weekly physical activity level between our groups suggests that independent-living older adults will seek out and perform their desired activity, in either a scheduled exercise program or other leisure-time activities. Also, the best predictor of current physical activity level in independent-living older adults was the activity performed over the past year.