Frank W. Booth, Ph.D. - US grants

immobilization of body part; neuromuscular disorder; molecular pathology; muscle disorders; striated muscles; amidases; deoxyglucose; glucose metabolism; obesity; glucocorticoids; calcium; genetic translation; exercise; hormone regulation /control mechanism; perfusion; cell free system; ribosomes;

Skeletal muscle will be from limbs that are rehabilitating from atrophy caused by a prior 7-day period of hindlimb immobilization in rats. The specific aims of the research plan are to: (1) Determine the synthesis rates of actin and cytochrome c in skeletal muscles at the 6th hr, 2nd day and 4th day of recovery from the prior 7-day immobilization. Synthesis rates are estimated in vivo by the constant infusion of 3H-tyrosine, measurement of the specific radioactivity of either purified actin or purified cytochrome c, measurement of the specific radioactivity of tyrosyl-tRNA and calculation of the estimated rate of fractional synthesis per day; (2) Determine the role of anabolic steroids and of various exercises (treadmill running, weight lifting and electrical stimulation) in addition to "normal" cage activity on the synthesis rates of actin and cytochrome c in skeletal muscles on the 2nd day of recovery from the prior 7-day period of hindlimb immobilization; (3) Determine the threshold of exercice duration needed to increase the synthesis rate of actin and cytochrome c in skeletal muscle on the 2nd day of recovery from a prior 7-day period of hindlimb immobilization; (4) Determine the sensitivity of increasing exercise duration on the amount of increase in actin or cytochrome c synthesis rates; (5) Determine the content of Alphaactin mRNA and of cytochrome c mRNA in skeletal muscles at the 6th hr, 2nd day, and 4th day of recovery from a prior 7-day period of hindlimb immobilization. The content of Alphaactin mRNA or cytochrome c mRNA will be semiquantitated by dot hybridization with cDNA for Alphaactin mRNA from rat skeletal muscle and a genomic clone of rat liver cytochrome c, respectively; and (6) Determine the content of Alphaactin mRNA and cytochrome c mRNA in skeletal muscles in the 3rd and 24th hours after an exercise bout when exercise is preformed on the 2nd day of recovery from a prior 7-day period of limb immobilization.

A long-term aim is to delineate the mechanisms by which chronic changes in contractile activity alter the phenotypic expression of skeletal muscle. In the regrowth of of atrophied skeletal muscle after ending limb immobilization, all proteins increase in response to normal muscle usage. Further increases in all proteins in normal adult skeletal muscles are only possible if it undergoes maximal loading exercise, such as weight-lifting. In contrast, repetitive, low-intensity exercise like running results in no enlargement of normal muscle, but causes an preferential expression of mitochondrial proteins, such as cytochrome c. A strategy of the present application is to compare the responses of mrna levels, protein synthetic machinery and potential molecular signals controlling the upregulation of specific protein quantities in different types of exercises known to cause various phenotypic expressions. The following methods will be employed in the comparison among exercises. In situ hybridization will permit the localization of transcripts to nuclei by using intron probes, so a determination can be made as the time when changes in the levels of specific nuclear transcripts occur after the exercise and for which transcripts. The four cytochrome c mRNAs will be isolated from a cDNA library, cloned, sequenced and translated so as to provide information on the functional significance of shifts in their relative profiles which occur during regrowth from atrophy and during a program of repeated daily bouts of running. A rat genomic library will be screened for fragments containing 5' upstream and coding sequences for somatic cytochrome c, a skeletal actin, fast myosin-heavy-chain and rRNA for the purposes of obtaining intron probes for in situ hybridization. Also, the 5' stream sequences will be used for a further proposal to study regulatory sequences for gene expression. In accordance with this long-range plan, studies are initiated in the present application to identify potential candidates for signals which may interact,directly r indirectly through a cascade, with the 5' untranscribed regulatory region of the genome. The potential roles of the oncogenes, ATP, creatine phosphate, H+, and Ca++as the linkers from increased muscle usage to differential gene expression will be considered.

The long-term objective of this research is to investigate why there is a detrimental decrease in the synthesis rates of the mixture of all myocardial proteins as the heart ages. The immediate aims of this present project are: (1) to determine whether the synthesis rates of selected specific proteins (cytochrome c, actin, ribosomal proteins, collagen and alpha and beta myosin heavy chains) decrease during senescence. The rationale is that to determine the mechanism for the molecular control of gene expression in the aging heart, correlations must be made between the synthesis rates of specific proteins and the levels of their mRNAs. Aim (2) is to correlate levels of protein synthesis and mRNA for these specific proteins in adult (1-yr old rat) and senescent (2-yr old rat) hearts. To determine whether a decreased capacity for the synthesis of these proteins is responsible for the decline in their content with aging in the heart, physical exercise and thyroxine will be used as experimental tools to enhance their synthesis. Therefore, aim (3) will be to determine if daily treadmill running will increase cytochrome c synthesis rate and also alter its mRNA level and profile in cardiac muscle. Aims (4) and (5) will be to determine if there is any defect in the capacity of the senescent heart to synthesize specific proteins and to increase the levels of their mRNAs in response to thyroxine. Aim (6) correlates the relationships between left ventricular pressure and dp/dt with the synthesis rates of specific proteins, specific mRNAs, age, thyroxine treatment and exercise. The synthesis rates of these proteins will be measured by the constant infusion of 3H leucine into unanesthesized rats in vivo. Specific mRNA levels will be determined in cardiac muscle by dot hybridization techniques using specific cDNA probes. Acute left ventricular cannulae will access pressures. This study will determine whether correlations exist between left ventricular pressures, aging, and factors (thyroxine and exercise) which cause volume overloading of heart and changes in the synthesis rates and mRNA levels of specific myocardial proteins. From such observations, future studies into the regulation of gene expression would be justified to determine the cause of the age- induced impairment in heart pumping capacity.

A long-term aim is to delineate the mechanisms by which chronic changes in contractile activity alter the phenotypic expression of skeletal muscle. In the regrowth of of atrophied skeletal muscle after ending limb immobilization, all proteins increase in response to normal muscle usage. Further increases in all proteins in normal adult skeletal muscles are only possible if it undergoes maximal loading exercise, such as weight-lifting. In contrast, repetitive, low-intensity exercise like running results in no enlargement of normal muscle, but causes an preferential expression of mitochondrial proteins, such as cytochrome c. A strategy of the present application is to compare the responses of mrna levels, protein synthetic machinery and potential molecular signals controlling the upregulation of specific protein quantities in different types of exercises known to cause various phenotypic expressions. The following methods will be employed in the comparison among exercises. In situ hybridization will permit the localization of transcripts to nuclei by using intron probes, so a determination can be made as the time when changes in the levels of specific nuclear transcripts occur after the exercise and for which transcripts. The four cytochrome c mRNAs will be isolated from a cDNA library, cloned, sequenced and translated so as to provide information on the functional significance of shifts in their relative profiles which occur during regrowth from atrophy and during a program of repeated daily bouts of running. A rat genomic library will be screened for fragments containing 5' upstream and coding sequences for somatic cytochrome c, a skeletal actin, fast myosin-heavy-chain and rRNA for the purposes of obtaining intron probes for in situ hybridization. Also, the 5' stream sequences will be used for a further proposal to study regulatory sequences for gene expression. In accordance with this long-range plan, studies are initiated in the present application to identify potential candidates for signals which may interact,directly r indirectly through a cascade, with the 5' untranscribed regulatory region of the genome. The potential roles of the oncogenes, ATP, creatine phosphate, H+, and Ca++as the linkers from increased muscle usage to differential gene expression will be considered.

DESCRIPTION: (Adapted from the applicant's abstract and Specific Aims.) Atrophy of skeletal muscle in humans begins in the third decade of life, but its functional significance is not realized until many decades later (when elderly people have reductions in skeletal muscle mass and strength below that required for independence in daily activities).The mechanisms of the aging- associated atrophy of skeletal muscle have not been established.However, strength training, such as weight lifting, can prevent the loss of muscle mass between the ages of 50-70 years (before senescence) in humans. A component of strength training, eccentric exercise (lengthening contractions), increases insulin-like growth factor-I (IGF-I) immunoreactivity within muscle fibers of young rats. Increases in IGF-I peptide and mRNA have been shown to be associated with muscle hypertrophy in tissue culture and in the work overload model, respectively. Conversely, IGF-I mRNA is decreased in skeletal muscle of young rats when the muscle was not allowed to support the load of body weight (which could be analogous to the decreased mobility or activity by older people). Thus, a potential relationship between weight-bearing exercise, IGF-I expression, and muscle size is suggested. Four Specific Aims will be tested. First, is the IGF system different between senescent and adult muscle? Does aging reduce the IGF system concurrent with atrophy of skeletal muscle? Measurements include mRNAs for IGFs and insulin- like growth factor binding proteins (IGFBPs), secretion of IGFs and IGFBPs from muscle, and receptor binding densities for IGF and growth hormone on muscle. Second, does the IGF system respond to a given bout of exercise differently between adult and senescent rats? Can eccentric exercise increase the IGF system in senescent muscle?Third, does senescent muscle have an insufficient IGF system to allow major regrowth from prior atrophy? Fourth, can the implantation of neonatal satellite cells, either with or without constitutive expression of IGF-I, into senescent muscle reverse the atrophy of senescent muscle? Does it take IGF-I peptide alone, or eccentric exercise alone, or both reverse atrophy in fast-type skeletal muscle between the ages of 21-24 months in Fischer 344 rats? These Specific Aims may help determine whether causal relationships exist between reductions in muscle activity, changes in IGF expression/modulation, and muscle atrophy in senescent rats. The potential significance of this application is that alterations of the IGF- I system will be tested as a possible mechanism for the atrophy of skeletal muscle with aging.

A long-term aim is to delineate the mechanisms by which chronic changes in contractile activity alter the phenotypic expression of skeletal muscle. In the regrowth of of atrophied skeletal muscle after ending limb immobilization, all proteins increase in response to normal muscle usage. Further increases in all proteins in normal adult skeletal muscles are only possible if it undergoes maximal loading exercise, such as weight-lifting. In contrast, repetitive, low-intensity exercise like running results in no enlargement of normal muscle, but causes an preferential expression of mitochondrial proteins, such as cytochrome c. A strategy of the present application is to compare the responses of mrna levels, protein synthetic machinery and potential molecular signals controlling the upregulation of specific protein quantities in different types of exercises known to cause various phenotypic expressions. The following methods will be employed in the comparison among exercises. In situ hybridization will permit the localization of transcripts to nuclei by using intron probes, so a determination can be made as the time when changes in the levels of specific nuclear transcripts occur after the exercise and for which transcripts. The four cytochrome c mRNAs will be isolated from a cDNA library, cloned, sequenced and translated so as to provide information on the functional significance of shifts in their relative profiles which occur during regrowth from atrophy and during a program of repeated daily bouts of running. A rat genomic library will be screened for fragments containing 5' upstream and coding sequences for somatic cytochrome c, a skeletal actin, fast myosin-heavy-chain and rRNA for the purposes of obtaining intron probes for in situ hybridization. Also, the 5' stream sequences will be used for a further proposal to study regulatory sequences for gene expression. In accordance with this long-range plan, studies are initiated in the present application to identify potential candidates for signals which may interact,directly r indirectly through a cascade, with the 5' untranscribed regulatory region of the genome. The potential roles of the oncogenes, ATP, creatine phosphate, H+, and Ca++as the linkers from increased muscle usage to differential gene expression will be considered.

DESCRIPTION (Applicant's abstract): How aerobic exercise signals skeletal muscle cells to increase mitochondria has been a question of interest for decades to many exercise scientists, including the applicant. Decreases in mitochondria occur in skeletal muscles of non-insulin dependent diabetes mellitus, obese, elderly, and physically inactive individuals and contribute to a reduced endurance to work in these populations. We recently found that muscles with a high mitochondrial content have little protein interaction with the 3'- untranslated region (UTR) of cytochrome c mRNA, compared to muscles with a low mitochondrial concentration. We also recently found a decreased RNA-protein interaction in muscles that were stimulated to increase cytochrome c mRNA. We believe this RNA-protein interaction is important for cytochrome c expression. The purposes of this proposal are to further characterize the structural and functional basis of the RNA-protein interaction and isolate the trans-proteins responsible for the regulation of cytochrome c gene. Specific aim 1 is to determine the sequence and structural basis for RNA-protein interaction is a 50-nucleotide (nt) region of the 3'-UTR of cytochrome c mRNA. A putative stem-loop structure, within which a 13-nt sequence was found to be conserved in the 3'-UTRs of rat, murine, human and chicken cytochrome c genes, was previously predicted by computer analysis within the 50-nt protein binding region. Specific aim 2 is to determine the function of RNA-protein interaction in the 3'-UTR of cytochrome c mRNA on its expression. Increased contractile activity could enhance cytochrome c gene expression by mRNA stabilization (which could be determined by measurement of its decay), or by translation acceleration (which could be determined by in vitro transcription and translation system). Specific aim 3 is to isolate and characterize the protein(s) binding to the 3'-UTR of cytochrome c mRNA. This information will provide information for identification of the signal that initiates cytochrome c adaptation to aerobic training. This proposal will contribute to an understanding of signaling mechanisms between exercise and mitochondrial biogenesis.

DESCRIPTION (Adapted from the applicant's abstract): The strategy of this proposal is to use a retrograde approach by starting at the actin promoter and tracing the signaling pathway back to the stretch stimulus. Serum response factor (SRF) binds to serum response element1 (SRE1) in the chicken skeletal actin promoter. SRE1 is necessary and sufficient for the increase in actin promoter activity caused by stretch of the anterior latissimus dorsi (ALD) muscle of young chickens. A faster migration of the SRF-SRE binding complex occurs from nuclear extracts of 3- and 6-day stretched ALD muscles, which indicates a post-translational modification to SRF. Specific aim 1 is to determine whether the phosphorylation status of SRF is altered during the stretch of chicken primary myocytes. 32P incorporation will be measured in SRF from stretched cells. Times of initial increases among SRF phosphorylation, skeletal actin mRNA, actin promoter activity, and faster migration of SRE-SRF in non-denaturing gels will be compared to determine the sequence of events. Precedence for the hypotheses in this proposal is provided by the extensive literature about the growth factor transactivation of c-fos by phosphorylation of SRF. Specific aim 2A is to determine which serine(s)/threonine(s) in the SRF is phosphorylated during stretch by the methods of two-dimensional phosphopeptide mapping and phosphoamino analysis. Precedence for specific aim 2 is that growth factors phosphorylate SRF and its accessory protein during induction of the c-fos promoter. Specific aim 2B will determine which nuclear protein kinase is responsible for phosphorylating SRF during stretch in cultured cells. Preliminary data show that stretch is associated with a faster mobility of SRF from nuclear but not cell extracts. Specific aim 3 will use the GAL4 system to determine the function of SRF amino acids which are phosphorylated by stretch. GAL4-SRF fusion proteins will demonstrate the region of SRF which transmits the stretch signal to the actin promoter. A key experiment will then be whether a site-specific mutation to an amino acid that is phosphorylated by stretch in the functional region of SRF abolishes the stretch-induced activation of SRF in the GAL4 system. This experiment's results will then be extended to the ALD muscle of young chickens. Specific aim 4 will use a dominant negative mutant of the kinase which was identified as responsible for stretch-induced activation of SRF. The molecular basis of muscle hypertrophy will assist physical training and pharmaceutical strategies to build skeletal muscle mass in the elderly for the purpose of keeping elderly out of nursing homes by maintaining their physical strength and thus their independence.

DESCRIPTION (Adapted from the applicant's abstract): Significant evidence suggests that skeletal muscle lipoprotein lipase (LPL) activity is a major determinant of triglyceride and lipoprotein metabolism. It is well documented that exercise training increases skeletal muscle LPL activity, decreases plasma triglycerides, increases HDL-cholesterol, and reduces the incidence of coronary artery disease. The long-term objective of the proposed investigations is to determine, at the level of the LPL gene, the mechanisms(s) by which exercise training increases skeletal muscle LPL mRNA expression. The first aim of the proposed studies is to systematically establish the time course, exercise duration, and muscles that LPL mRNA is increased in run-trained rats. It is hypothesized that during exercise training, LPL mRNA increases after each exercise session to peak several hours after exercise and then gradually falls to untrained sedentary levels within 24-48 hours of rest. It is also hypothesized that 10 min. of intense running or electrical stimulation of the motor nerve is sufficient to increase skeletal muscle LPL mRNA. These studies will measure the transcription rate of LPL in skeletal muscles of run trained and sedentary control animals. Deletion and mutation experiments on the LPL promoter will determine essential and necessary gene sequences for exercise induced LPL promoter regions. Alternative experiments are planned to test for the involvement of the 3'-untranslated region of LPL in causing the exercise-induced increase in LPL expression. These findings will elucidate the molecular mechanism of increased LPL mRNA levels during exercise training, and thus will be essential for identifying the biochemical signaling pathway(s) involved in exercise increased LPL expression and improved blood lipids. These specific aims are at the interphase of linking molecular mechanisms regulating LPL expression to the prevention of morbidity and mortality caused by coronary artery disease, the leading cause of death in the United States.

DESCRIPTION (Adapted from the applicant's abstract):Promoting skeletal muscle growth is extremely important for medical conditions in which muscle wasting contributes to low quality of life, high health care costs, and institutionalization (i.e. aging, cachexia, congestive heart failure, etc.). It is well known that skeletal muscle hypertrophies in response to increased loading; however the mechanisms underlying this phenomenon remain poorly understood. In this respect, there is accumulating evidence that hormonal signaling and mechanical signaling (via focal adhesion complex proteins) may act synergistically and share common pathway intermediates such as focal adhesion kinase (FAK), serum response factor and other upstream/downstream elements. To examine this possibility, the current proposal consists of a strategy to tease out the individual and combined effects of angiotensin II signaling, insulin-like growth factor-I (IGF-1) signaling and integrin (mechanical) signaling in skeletal muscle hypertrophy. In the first specific aim, the PI proposes to confirm and extend upon two pilot experiments in which, using an overload model (via synergistic gastrocnemius ablation), compensatory hypertrophy of the rat soleus muscles is almost completely inhibited by the use of an ACE inhibitor. Although angiotensin II is a known stimulus to cardiac and smooth muscle growth, these findings in skeletal muscle are novel. The second specific aim consists of multiple experiments designed to a) in living rats, test the effect of angiotensin II alone, IGF-1 alone, and mechanical loading alone (rat resistance exercise model) on the acute (1 hr post stimulus) response of multiple downstream signaling elements common to these 3 stimuli in skeletal muscle; b) in living rats, test the effect of skeletal muscle loading on these downstream elements while either blocking endogenous angiotensin II input (receptor blockade or ACE inhibition), blocking endogenous IGF-1 input (receptor blockade), or blocking mechanical signaling (disrupting the integrin/extracellular matrix interface with RGD peptide); and, c) in cultured rat skeletal myotubes, test the effect of exogenous angiotensin II alone, exogenous IGF-1 alone, and stretch alone on these downstream elements. Finally, the third specific aim utilizes microarray technology to examine the overlap between these growth factor and mechanical stimuli at the level of mRNA expression in skeletal muscle of living rats. Elucidation of the mechanisms signaling skeletal muscle growth may lead to drugs, gene therapy, or other countermeasures against skeletal muscle wasting in individuals for whom exercise is perhaps impossible.

DESCRIPTION (Adapted from the applicant's abstract): Satellite cells are muscle-specific stem cells that function to repair damaged myofibers and provide new myonuclei for muscle enlargement. Rosenblatt has shown that knocking out the proliferative capacity of satellite cells prevents hypertrophy of skeletal muscle. Blau and Wright have found that satellite cells prematurely senesce in young patients with Duchenne's muscular dystrophy who have many cycles of regeneration. Schultz has observed a progressive loss of the proliferative capacity of satellite cells as rats age, and similar data has just been reported in humans. Hayflick showed that normal, diploid cells have a finite proliferative lifespan and reach cellular senescence. However, Bischoff indicated that a critical evaluation of the self-maintenance criteria required to categorize the satellite cell as stem cell is yet to be undertaken. These provocative reports highlight some of the conceptual framework to pose the following specific aims. Using the well-established and validated approach of clonogenecity assays to determine a cell's proliferation potential, this proposal examines 1.) whether a physiological model of repeated cycles of atrophy-regrowth in old skeletal muscle speeds satellite cells to senescence so that their proliferative capacity is depleted prior to the lifespan of rats; 2.) determine whether the application of IGF-1 to skeletal muscle or 3.) increased contractile activity, or both aims 1 and 2 results in either a.) using up or b.) replenishing the finite population doublings in old satellite cells. These results would shed much needed insight into whether replicative senescence can be modulated by environmental factors. The information thus gleaned from these studies will provide the basis for follow-up experiments that will measure cell cycle markers to begin to explain the observations in molecular detail. As the number of individuals with frailty is rapidly increasing, it becomes a more urgent clinical, social, and economic issue to find out if and how satellite cell lifespan can be maintained/enhanced. This proposal will therefore provide novel insights into how the self-maintenance properties of satellite cells is modulated by compensatory factors (IGF-1 and exercise), thereby forming the basis for more effective interventions against senile-atrophy and frailty.

striated muscles; atrophy; biomedical resource;

DESCRIPTION (provided by applicant): Insulin resistance and type 2 diabetes are epidemic in adults, and are now even occurring in adolescents. A decrease in physical activity has played an important role in this increase in diabetes as documented in many epidemiological and physiological papers. Of great significance are recent publications showing that increased contractile activity signals an enhanced glucose uptake through an insulin-independent signaling pathway, likely AMP kinase, but the complete pathway remains to be delineated. The importance of these observations is that they raise the probability that unexpected novel proteins linking physical inactivity to insulin resistance will be found. As the post-genome era begins with the sequencing of the human genome, tools are now available to discover the identity of proteins currently unassociated with the signaling of insulin resistance by mechanisms other than insulin modification. This proposal focuses on those proteins differentially expressed when either normal voluntary running ceases due to the removal of a running wheel from the cage, or when high fat diets are consumed. These models mimic current lifestyles of sedentary activity and/or high fat consumption. Specific aim I will use 2-D gel electrophoresis to experimentally determine differentially expressed proteins in skeletal muscle that have undergone decreased physical activity. Specific aim 2 will also employ 2-D gel electrophoresis to determine differentially expressed proteins in the skeletal muscle of rats that have undergone decreased physical activity while eating a high fat diet. One hypothesis is that both inactivity and high blood lipids will cause unique, but not identical, sets of proteins related to insulin resistance to be expressed in skeletal muscle. Many of these proteins will heretofore be unidentified as playing a role in skeletal muscle insulin resistance. Identifying the expressed proteins associated with insulin resistance in skeletal muscle will permit the development of new hypotheses, whose functions and interactions with other proteins will be the focus of future grant applications. Such new hypotheses could lead to new therapies against diabetes. Outcomes of this proposal will better establish that healthy active skeletal muscles interact with other organ systems to prevent the metabolic disorders of type 2 diabetes, atherosclerosis, and obesity.

The U.S. now ranks 26th out of 35 for overall life expectancy among other developed and developing countries. PROBLEM: Sedentary lifestyle increases the risk of 40 chronic diseases. Most (?97%) adults do not meet U.S. guidelines for weekly physical activity, thereby increasing the onset of chronic disease, beginning at a younger age. Understanding the biological proclivity for sedentary living could help identify interventions to increase U.S. life expectancy. Growing evidence suggests that genetics plays a significant role in decreasing the motivation to be physically active. For example, in 772 mono- and di-zygotic twins, data was published showing sedentary behavior is moderately heritable in adults based upon between-twin variation in sedentary behaviors. PRELIMINARY STUDIES: Rats were successfully bred for the phenotype of low voluntarily wheel- running (LVR) motivation and potential gene candidates specific to the nucleus accumbens, a region integral to motivated behaviors, were found. One candidate gene, protein kinase inhibitor alpha (Pki?), was shown to be lowly expressed in LVR compared to outbred, wild-type (WT) rats and was further addressed due to its key position in modulating motivation-related signaling. Local overexpression of the Pki? in the nucleus accumbens of female LVR rats increased voluntary running distances 3.1-fold compared to pair-matched empty vector controls. Amazingly, WT rats did not increase nightly running distance following Pkia overexpression and appeared to show molecular resistance to Pki? overexpression. OBJECTIVES: A better understanding of mechanisms that control the lack of motivation for physical activity behavior can help facilitate the development of potential drug therapies for sedentary individuals and combat age-related decreases in physical activity over the lifespan. As such, the goal of this application is to expand upon these findings and help reverse sedentary lifestyle. STRATEGY: Aim 1 will further test the ability of Pki? overexpression in the nucleus accumbens of wild-type (WT) and LVR rats at 15 and 24 weeks of age to increase voluntary running behavior and the molecular responses in male and female rats. Two hypotheses are that Pki? overexpression in nucleus accumbens will increase voluntary running distance: 1) in young LVR rats, but not WT because of their differences in molecular responses; and 2) in both LVR and WT 24-wk-old rats whose running started less than their younger counterparts. Aim 2 will examine whether differences in either single-nucleotide polymorphisms, methylation, and/or differentially bound transcription-factors exist in the promoter of LVR vs. WT rats in 15- and 24-wk-old rats. The outcome could help to explain the differential expression of Pki? in different rat models that correlate with different voluntary activity behaviors. SIGNIFICANCE: This 2-yr R21 project will advance our understanding of molecular mechanisms responsible for sedentary behavior associated with aging and genetic background. Pki? rescue of running from low voluntary running sets guides for potential therapies to lengthen the desire for vigorous physical activity into old age, thereby increasing health and longevity.

PROJECT SUMMARY/ABSTRACT Physical inactivity is linked to at least 40 chronic disease conditions including insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, advanced brain aging, loss of cognition, and neurodegeneration. In contrast, regular exercise and maintenance of higher cardiorespiratory fitness expands health-span by maintaining each of these factors and reducing risk for a myriad of chronic conditions. While the beneficial effects of exercise are extensively recognized, the molecular mechanism(s) underpinning these benefits are less well understood. Existing literature and data recently released by MoTrPAC indicate that liver-derived factors may play a central role in the systemic benefit of exercise. Studies in this proposal will profile known and unknown factors released from the liver after exercise that may serve as molecular transducers of exercise and drive positive adaptations in liver, skeletal muscle, and brain health. We have focused on MoTrPAC data revealing a robust ~200-fold acute exercise induced upregulation in hepatic mRNA expression of orphan nuclear receptor neuro-derived clone 77 (Nur77 or NR4A1) that occurred in conjunction with an elevation in hepatic fibroblast growth factor 21 (FGF21) mRNA and elevated plasma ketone levels. Nur77 and FGF21 are intimately linked, as Nur77 transcriptionally regulates FGF21 and both are known to regulate hepatic ketogenesis. In addition, both FGF21 and ketone bodies are primarily liver-derived and both are known to have strong systemic and neuroprotective properties. However, little is known about the role of these factors in exercise-mediated changes in brain and cognitive health. Here we will test our central hypothesis that exercise-induced hepatic adaptations are central to the molecular adaptations that occur in the skeletal muscle and brain with acute and chronic exercise. We will mechanistically interrogate if hepatic Nur77 (via a liver-specific AAV-shRNA knockdown approach) is a critical exercise-induced factor driving both hepatic FGF21 and ketone production in male and female Fischer 344 rats (Aim 1). Similarly, we will target ketogenesis directly by knocking down liver HMG-CoA synthase 2 (HMGCS2, the key regulatory enzyme in hepatic ketogenesis) (Aim 1). In addition, we will perform an unbiased screen of extracellular vesicles and miRNAs released by the liver in response to acute and chronic exercise training and test whether there are novel secreted factors originating in the liver regulate carbohydrate and lipid metabolism in neuronal and skeletal muscle cells (Aim 2). Collectively, our proposed approaches will establish the critical mechanistic importance of Nur77 and HMGCS2 in the regulation of hepatic FGF21 and ketone-mediated benefits in liver, skeletal muscle, and brain health. In addition, these studies will also potentially identify other novel exercise-induced molecular transducers originating from the liver.