Lisa A. Lesniewski, Ph.D. - US grants

DESCRIPTION (provided by applicant): Type 2 diabetes and the metabolic syndrome are epidemic in the US, and a hallmark of both is insulin resistance. Understanding the mechanisms of insulin action is essential in the treatment of these disorders. Recently, a signaling pathway has been identified that links insulin stimulation of its receptor to glucose transport independent of PI3K. This pathway involves the activation Cbl and its adaptor protein CAP and is known to stimulate glucose uptake in cultured adipocytes. In addition to a role in stimulating glucose uptake, the Cbl/CAP complex is involved in the proliferation and motility of macrophages. As macrophage activation and chronic inflammation have been associated with insulin resistance, this dual role of Cbl/CAP may have important consequences. The general purpose of this proposal is to examine the role of CAP in regulating in vivo insulin action. The specific aims are 1) to determine if whole body gene knockout of CAP results in alterations in insulin action, 2) to determine if CAP knockout results in an upregulation of the alternate PI3K-dependent signaling pathway, and 3) to determine if macrophage infiltration, activation and cytokine production is altered in CAP knockout mice and is related to the putative changes in insulin action.

DESCRIPTION (provided by applicant): The candidate, Lisa A. Lesniewski Ph.D., is a physiologist in the Department of Integrative Physiology at the University of Colorado at Boulder. Dr. Lesniewski's research focuses on the mechanisms mediating vascular and metabolic dysfunction with aging and how habitual aerobic exercise can preserve physiological function with aging. Her immediate goal is to acquire new research and professional skills that will help her to achieve her long-term career goal of developing a successful independent, extramurally-funded research program on the prevention and treatment of vascular and metabolic aging. The proposed KO1 award will provide Dr. Lesniewski the necessary support to achieve this goal. CAREER DEVELOPMENT PLAN. Dr. Lesniewski's research career development training activities will consist of: 1) acquiring new research skills associated with and complementary to the proposed research plan;and 2) structured activities including formal course-work, attendance and presentations at weekly journal clubs, university seminar series and scientific meetings, and regular interactions with her mentoring team. ENVIRONMENT. The environment for Dr. Lesniewski's training should be outstanding. The sponsor, Dr. Douglas Seals, is a well-established extramurally-funded scientist with a strong record of successful mentoring in biomedical aging research. He is complemented by a group of consulting mentors that consist of internationally- recognized investigators, each providing specific expertise in a key area of Dr. Lesniewski's research project and overall training plan. RESEARCH. The aims of the research project are to establish if aging increases susceptibility to the deleterious effects of a high fat, or Western diet (WD) by exacerbating chronic low grade inflammation and to determine if habitual exercise can prevent the deleterious effects of WD on vascular and metabolic function by reducing inflammation. The proposed research will use an "integrative" (system to gene) approach in young, middle-aged, and older mice to identify the cellular and molecular mechanisms by which WD causes inflammation-associated endothelial and metabolic dysfunction with aging and regular exercise exerts protective effects. The results should provide clinically important information regarding: 1) the influence of aging in altering vulnerability to a commonly encountered factor, WD and 2) the mechanisms by which WD causes and habitual exercise prevents these deleterious effects. PUBLIC HEALTH RELEVANCE: Advancing age and consumption of a WD are associated with vascular and metabolic dysfunction and disease. However, it is unknown if the adverse effects of WD become greater with aging, the mechanisms by which this occurs, and if these effects can be prevented by habitual aerobic exercise.

DESCRIPTION (provided by applicant): Advancing age is associated with the development of vascular dysfunction and disease. However, the mechanisms involved are incompletely understood. One novel and largely unexplored hypothesis is that "physiological resistance" to the adverse effects of common environmental factors to which we are chronically exposed decreases with aging, thus exacerbating the resulting dysfunction and increased risk of disease. One such factor may be a "Western", i.e., high-fat, diet (WD). The overall goal of this project is to examine the mechanisms by which WD exacerbates age-associated vascular endothelial dysfunction. Specifically, we will examine the effect of WD on signaling pathways involved in the regulation of vascular inflammation, oxidative stress and apoptosis in middle-aged (MA) and older (O) mice. The specific aims are (1) to measure endothelium dependent dilation (EDD) and nitric oxide (NO) bioavailability in the carotid arteries of MA/O mice, to determine if advancing age is associated with a pro- inflammatory, pro-oxidative, pro-apoptotic phenotype, (2) to determine if WD increases the activation of the pro-inflammatory and apoptotic signaling molecules;nuclear factor kappa B (NFkB), c-jun NH2 terminal kinase (JNK), and forkhead box O (FoxO) in MA/O mice and (3) to determine if inhibition of NFkB or JNK can reverse WD-associated vascular dysfunction, inflammation, oxidative stress and apoptosis in MA/O mice. To do so, we will study young (Y: 6-8 mo), MA (18-20 mo) and O (30-32 mo) male B6D2F1 mice. Mice will be fed standard chow (NCD, 12% kcal from fat) or a custom WD (40% kcal from fat). Vascular endothelial function will be measured in isolated carotid arteries. Nitric oxide bioavailability and activation of NFkB, JNK and FoxO will be assessed in aortic lysates. Lastly, we will utilize pharmacological inhibition of NFkB and JNK to determine their roles in WD-associated vascular endothelial dysfunction, inflammation, oxidative stress and apoptosis in MA/O mice. The expected results will provide novel insight into the mechanisms by which WD exacerbates age-associated vascular dysfunction. PUBLIC HEALTH RELEVANCE: Advancing age and consumption of a WD are associated with vascular dysfunction and disease. This proposal aims to determine if the adverse effects of WD become greater with advancing age and the mechanisms by which this may occur.

DESCRIPTION (provided by applicant): Cardiovascular disease (CVD) is the leading cause of death in the United States. Aging is the major risk factor for development of CVD. The major age-related arterial phenotypes, which are thought to be responsible for the development of CVD in older adults, are reduced endothelial function and enhanced large artery stiffness. Recently, it has been demonstrated that inhibition of the mTOR signaling pathway, via rapamycin or genetic manipulation, extends lifespan and reduces age-related physiological dysfunction. The proposed studies aim to determine (a) if increased mTOR signaling is responsible for age-related arterial dysfunction and (b) whether dietary rapamycin treatment and activation of AMPK, a putative rapalog, can reverse age-related arterial dysfunction. Our laboratory has performed preliminary studies suggesting that arterial activation of mTOR is increased with advancing age and that this is concomitant with age-associated endothelial dysfunction and large artery stiffening known to result from increases in oxidative stress and inflammation. Furthermore, dietary treatment of old mice with mTOR inhibitor, rapamycin, can reverse this age-associated arterial phenotype and improve arterial function. In the present application, by utilizing dietary rapamycin treatment, we will directly assess the role that mTOR plays in modulating transcription factor activity and downstream gene/protein expression and the subsequent effects on arterial phenotype and function. In addition, we will determine if pharmacological activation of AMPK by AICAR will act as a rapalog, mimicking the effects of rapamycin-induced mTOR inhibition on arterial function and phenotype.

? DESCRIPTION (provided by applicant): Advancing age is the primary non-modifiable risk factor for cardiovascular diseases (CVD), such as atherosclerosis, and arterial dysfunction is an important contributor to this increased disease risk. The proposed studies will explore the novel hypothesis that an age-associated reduction in microRNA, miR-92a and its cluster miR-17-92, underlie age-associated arterial dysfunction and accelerated atherosclerotic disease progression. Because obesity is epidemic in US adults and is often a co-morbidity of older age, we will study both normal chow and high fat fed young and old mice to determine if aging per se exaggerates the negative influence of diet-induced obesity on vascular function and disease and if this is mediated by further decreases in arterial miR-92. We will utilize the well-established B6D2F1 mouse model of vascular aging and manipulate miR-92 by in vivo treatment with inhibitor and mimic oligomers as well as utilize genetic models to both up - and down-regulate miR-17-92, in the endothelium and whole body; respectively. We will assess measures of arterial function, such as endothelium dependent dilation, angiogenesis and large artery stiffening as well as assess atherosclerotic disease progression using a surgical model of disturbed carotid artery blood flow in mice crossed into an athero-prone background. Using targeted and unbiased approaches, we will explore the downstream targets of miR-92a/miR-17-92 and how these impact arterial function and disease. Last, using in vitro cell and tissue culture models we will determine upstream modulators of miR-17-92 expression, including regulation by the transcription factors c-myc and p53, as well as the role of altered shear stress in the regulation of miR-17-92 expression. The results will elucidate a novel mechanism underlying age-associated vascular dysfunction and disease risk and will provide critical evidence for a novel therapeutic target that may be effective in the treatment of CVD in older adults.

? DESCRIPTION (provided by applicant): Advancing age is the primary non-modifiable risk factor for cardiovascular diseases (CVD), such as atherosclerosis, and arterial dysfunction is an important contributor to this increased disease risk. The proposed studies will explore the novel hypothesis that an age-associated reduction in microRNA, miR-92a and its cluster miR-17-92, underlie age-associated arterial dysfunction and accelerated atherosclerotic disease progression. Because obesity is epidemic in US adults and is often a co-morbidity of older age, we will study both normal chow and high fat fed young and old mice to determine if aging per se exaggerates the negative influence of diet-induced obesity on vascular function and disease and if this is mediated by further decreases in arterial miR-92. We will utilize the well-established B6D2F1 mouse model of vascular aging and manipulate miR-92 by in vivo treatment with inhibitor and mimic oligomers as well as utilize genetic models to both up - and down-regulate miR-17-92, in the endothelium and whole body; respectively. We will assess measures of arterial function, such as endothelium dependent dilation, angiogenesis and large artery stiffening as well as assess atherosclerotic disease progression using a surgical model of disturbed carotid artery blood flow in mice crossed into an athero-prone background. Using targeted and unbiased approaches, we will explore the downstream targets of miR-92a/miR-17-92 and how these impact arterial function and disease. Last, using in vitro cell and tissue culture models we will determine upstream modulators of miR-17-92 expression, including regulation by the transcription factors c-myc and p53, as well as the role of altered shear stress in the regulation of miR-17-92 expression. The results will elucidate a novel mechanism underlying age-associated vascular dysfunction and disease risk and will provide critical evidence for a novel therapeutic target that may be effective in the treatment of CVD in older adults.