Abstract
Metabolic Regulation and Structural Evaluation of APOBEC-1 Complementation Factor
Chad A. Galloway, Ph.D.
University of Rochester, New York, 2009
APOBEC-1 complementation factor (ACF) is an AU-rich RNA-binding protein discovered through its requirement to properly target cytidine to uridine deamination of C6666 in apoB mRNA in the intestinal tissue of all mammals and the livers of select species. Metabolic perturbations that enhance editing efficiency in hepatic tissue, specifically insulin and ethanol stimulation, were correlated with the accumulation of phosphorylated nuclear ACF. Through ACF purification and phosphoamino acid analysis, I determined serine phosphorylation to be the metabolically regulated site(s). Inhibition of protein phosphatase 1 by cantharidin resulted in the nuclear retention of ACF and suggested that ACF’s nuclear export required its dephosphorylation. Treatment of primary rat hepatocytes with protein kinase activators suggested PKC as the kinase active on ACF and in vitro phosphorylation with recombinant PKC demonstrated effective phosphorylation of recombinant ACF, while PKA could not phosphorylate ACF. Metabolic labeling of primary hepatocytes from APOBEC-1 knockout mice and human primary hepatocytes demonstrated that ACF phosphorylation was not dependent on its interaction with APOBEC-1 (the cytidine deaminase targets to apoB mRNA for editing) nor was this post-translational modification of ACF species-specific. Significantly, in apoB editing incompetent tissue, human liver, the phosphorylation of ACF was on serine suggestive of a general role of phosphorylation in ACF regulation. Cumulative evidence supported an interaction of ACF with apoB mRNA in both the nucleus and cytoplasm. The hypothesis tested in this thesis is that ACF may have a function in the export of apoB mRNA from the nucleus to the cytoplasm. In this regard, ACF trafficking to the nucleus and back out to the cytoplasm may modulate the availability of apoB mRNA for ApoB protein translation. Evaluation of the hypothesis in the leptin-deficient Ob/Ob mouse model of obesity and insulin resistance demonstrated leptin regulation of hepatic ACF expression. In this animal model, ACF nuclear retention was not responsive to insulin though ACF phosphorylation was compartmentalized to the nucleus. The accumulation of cytoplasmic ACF in Ob/Ob mice correlated with an increased proportion of cytoplasmic apoB mRNA, relative to lean controls. In the cytoplasm, ACF was localized to the low-density microsomal (LDM) fraction (the site of apoB translation) in both mice with elevated levels observed in the Ob/Ob mice. This supported the hypothesis that ACF association with apoB mRNA may be functionally significant to apoB mRNA translation. I propose that the interactions of ACF with apoB mRNA and its role in hepatic trafficking of this mRNA may serve as a paradigm for a more global function of ACF. Similar to other members of the ELAV/Hu family of RNA-binding proteins, ACF may control the stability and trafficking of other transcripts and this might explain the ubiquitous tissue expression of ACF and its requirement for embryonic development. In light of this, I have defined a minimal functional portion of ACF, comprised of amino acids 1–320 (ACF320), capable of specifically binding apoB RNA and APOBEC-1. As such, ACF320 was sufficient to complement editing. Purification of recombinant ACF320 and analytical ultracentrifugation suggested the protein existed as a monomer in the absence of RNA. However, in the presence of RNA, multimeric complexes of ACF320 were observed. This observation was confirmed in vivo where ACF320 self-associated with the ACF subunits oriented with N-termini in close proximity as determined by FqRET analyses. The findings in this thesis will serve to lead investigation on the structure and function of ACF and facilitate future research to identify new mRNA targets of ACF, its role in post-transcriptional control of gene expression, and its regulation of development.
Dietary Polyunsaturated Fatty Acids and Highly Unsaturated Fatty Acids Differentially Affect the Development of Nonalcoholic Fatty Liver Disease
Whitney J. Sealls, Ph.D.
Albany Medical College of Union University, New York, 2008
Nonalcoholic fatty liver disease (NAFLD) is a medical term that encompasses both benign fatty liver and pathological nonalcoholic steatohepatitis (NASH), in the absence of excessive alcohol consumption. In the United States, NAFLD is the most frequent cause of abnormal serum liver enzymes and often positively correlates with other symptoms of the metabolic syndrome. Due to the increasing prevalence of humans with diseases encompassed by the metabolic syndrome, NAFLD is becoming an increasing problem in industrialized countries. Therefore, great importance is being given to identifying both the risk factors involved in the development of NAFLD and possible treatment modalities for those inflicted with the disease. Current treatments for NAFLD are underdeveloped and thus a great need for therapeutics exists.
Recent work has pointed to the importance of dietary nutrients, specifically lipids and lipid-regulated gene transcription in the development and treatment of NAFLD. Specifically, unsaturated fatty acids are emerging as key players in the lipid-regulated transcription involved in NAFLD. Much work regarding unsaturated fatty acids in NAFLD utilizes dietary studies in which multiple types of unsaturated fatty acids are grouped together. We believe that different unsaturated fatty acid classes play different roles in the disease process and thus should be separated within an experimental framework to deduce their individual effects. In our work, we specifically set out to look at the affects of different classes of fatty acids on the prevention of NAFLD.
We utilized a hepatic mouse knockout model, the liver-specific cytochrome P450 reductase knockout, that developed NAFLD by 8 weeks and that had an apparent hepatic defect in the ability to convert dietary polyunsaturated fatty acids (PUFAs: C18:2o6 and C18:3o3) into highly unsaturated fatty acids (HUFAs: C20:4o6, C20:5o3, C22:6o3). We hypothesized that the inability of these mice to make HUFAs was contributing to the NAFLD phenotype observed by altering transcriptional profiles. We also hypothesized that dietary supplementation with premade HUFAs would prevent the development of NAFLD.
To test our hypotheses, we utilized diets differing only in fatty acid composition. We found that nulls fed premade dietary HUFAs had a dampened NAFLD phenotype compared to nulls fed a diet rich in saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), or PUFAs. Nulls fed a diet rich in PUFAs developed severe steatosis comparable to nulls fed a diet rich in SFAs and MUFAs. Surprising was the finding that control animals, which do not have a defect in HUFA synthesis, fed a diet rich in PUFAs also developed steatosis similar to that of controls fed a diet rich in SFAs and MUFAs. This is a novel finding in that the current dogma is that PUFAs taken in by the diet are efficiently converted into HUFAs and that these de novo-synthesized HUFAs have the same effect as premade HUFAs. Our work highlights the key differences in PUFAs and HUFAs in the development of NAFLD. Our work suggests that: (1) dietary HUFAs fed to animals that develop NAFLD limits the hepatic storage of lipid by repressing lipogenic pathways controlled by the transcription factors, PPARg, SREBP1, and SREBP2 and (2) de novo-synthesized HUFAs, in contrast to dietary HUFAs, are unable to repress the same lipogenic pathways and thus hepatic lipid storage prevails. Although PUFAs/HUFAs are thought to positively regulate fatty acid oxidation genes in the fasted state through regulation of PPARa, we did not observe significant differences in this pathway by altering dietary lipid. Our work significantly contributes to the understanding of the differential hepatic affects of PUFAs and HUFAs on hepatic lipid storage and gene expression and highlights the importance of dietary intake of HUFAs and not PUFAs. The American Heart Association (AHA) currently recommends eating at least 2 fish meals per week as a source of o3 HUFAs. They also recommend eating precursor PUFAs and acknowledge that the beneficial effects of PUFAs versus HUFAs is both controversial and not well understood. Our work significantly contributes to the differential affects of PUFAs and HUFAs on a background of NAFLD.
Cardiovascular Disease Management and Functional Capacity in Patients With Metabolic Syndrome
Melissa D. Zullo, Ph.D.
Case Western Reserve University, Ohio, 2009
Hemodynamic Factors in the Insulin Resistance of Obesity and Hyperlipidemia
Jenny Dong-Nee Chiu, Ph.D.
University of Southern California, 2009
Insulin resistance is an important risk factor for the development of type 2 diabetes, obesity, cardiovascular disease, hypertension, and certain cancers. Although it is highly prevalent across the world, the pathophysiology remains unclear. Most studies concentrate on the defects observed at the target tissue, specifically at the cellular signaling level. However, before activating intracellular pathways in skeletal muscle, secreted insulin must first find its way through the vascular beds, cross the endothelial barrier, diffuse through the interstitial fluid, and bind insulin receptors. Although these steps are critical for insulin to act peripherally, these processes are poorly characterized. Intravenous insulin infusion rapidly increases plasma insulin, yet glucose disposal occurs at a much slower rate. This delay in insulin’s action may be related to the protracted time for insulin to traverse capillary endothelium. The purpose of the first study was to investigate whether bypassing the transendothelial insulin transport step and injecting insulin directly into the interstitial space would moderate the delay in glucose uptake observed with intravenous administration of the hormone.
To test this, I performed sequential intramuscular injections of saline (n = 3) or insulin (n = 10) administered directly into the vastus medialis of anesthetized dogs. Injections of 0.3, 0.5, 0.7, 1.0, and 3.0 units of insulin were administered hourly during a basal insulin euglycemic glucose clamp (0.2 mU/min/kg). These injections with 30-gauge needles served to deliver insulin directly into the interstitial fluid that bathes the myocytes, thereby bypassing the potentially rate-limiting step of insulin transport. Local glucose uptake in each leg was calculated based on arteriovenous glucose differences and Doppler-measured blood flow. Unlike the saline group, each incremental insulin injection caused interstitial (lymph) insulin to rise within 10 min, indicating rapid diffusion of the hormone within the interstitial matrix. Delay in insulin action was virtually eliminated, indicated by immediate dose-dependent increments in hind limb glucose uptake. Additionally, bypassing insulin transport by direct injection into muscle revealed a 4-fold greater sensitivity to insulin of in vivo muscle tissue than previously reported from intravenous insulin administration. These results indicate that the transport of insulin to skeletal muscle is a rate-limiting step for insulin to activate glucose disposal. Based on these results, one may speculate that defects in insulin transport across the endothelial layer of skeletal muscle will contribute to insulin resistance.
Elevated plasma levels of FFAs are often observed in obese and diabetic individuals and have thus been implicated as a causative link between obesity and insulin resistance. Although many have focused mainly on lipids’ effect to inhibit insulin signaling at the muscle cell, FFAs have several hemodynamic effects that may contribute to the development of insulin resistance. This second study was conducted to determine if insulin resistance, as mediated by elevated plasma FFAs, hinders transport of insulin to the interstitial space. I performed hyperinsulinemic euglycemic clamps (1 mU/min/kg) with saline infusion (n = 5, CON) or intralipid plus heparin (n = 6, INL) in lean, healthy mongrel dogs. The intralipid plus heparin infusion (20% fat emulsion at 1.5 mL/ min) served to create an acutely hyperlipidemic insulin-resistant dog model. Intralipid infusion did not change blood pressure or macrovascular (femoral artery) blood flow. Upon infusion, plasma FFA levels increased rapidly to a maximum level of 2.8 mM. Lymph FFAs rose much more slowly and were greatly attenuated increasing to a maximum of only 0.18 mM. Whole body glucose uptake rose from basal 3 to a steady state 7 mg/min/kg in the CON group, compared to a marked decline from maximum 6 to 4 mg/min/kg after 154 min of lipid infusion in the INL group. Endogenous glucose production was immediately suppressed in the CON group, while no suppression was observed in the INL animals. Interestingly, the time point when maximal FFA level in the interstitial fluid was achieved coincided with the time point glucose uptake began its decline. There was no significant difference in interstitial insulin concentrations between CON and INL groups (44.8 vs. 50.7 mU/L, respectively). However, when resistance was established (t = 130–360 min), lymph to plasma insulin gradient was significantly different (0.58 vs. 0.62; CON vs. INL, P < 0.001). In addition, there existed a strong correlation between lymph insulin and glucose uptake rates (r = 0.98) in the CON group, which was lost in the INL group (r = 0.71). These data suggest that there is rapid induction of resistance in the liver and peripheral tissues with elevated plasma free fatty acids. The transport of FFAs is temporally associated with reduction of glucose disposal. Although FFAs do not cause a decrease in interstitial insulin concentrations, increased lymph to plasma insulin gradient in the INL group suggests that more insulin was able to enter the interstitial space, suggestive of an increase in capillary permeability. The interstitial insulin in the INL group was not able to effectively increase glucose uptake, which may suggest resistance at the cellular level. However, due to the steady state, saturating nature of this study, a more dynamic approach to examine FFAs’ role in altering hemodynamic transport of insulin is warranted.
In this next study, a nonsaturated approach to determine whether FFAs hinder transport of insulin was examined. I tested the effect of insulin resistance induced by hyperlipidemia on the dynamics of insulin injected into skeletal muscle. The injections serve to bypass the endothelial barrier that if is truly rate limiting, then insulin sensitivity would be rescued and glucose uptake would be comparable to control animals. Basal insulin euglycemic clamps (0.2 mU/min/kg) with or without lipid infusions (20% at 1.5 mL/min) were performed on anesthetized dogs. Similar to the first study, sequential insulin doses were administered by intramuscular injection directly into the vastus medialis of one hind leg, using the contralateral leg for comparison.
Intramuscular insulin injection in normal animals (n = 10) caused a clear dose-dependent increment in interstitial insulin levels, as well as dose-dependent increase in leg glucose uptake. In a second group of animals (n = 8), lipid was infused before and during intramuscular insulin injection to cause systemic increase in free fatty acids. In sharp contrast, systemic lipid infusion caused insulin resistance, indicated by reduced glucose infusion required to maintain euglycemia, and prevented injection-induced increase in lymphatic insulin and leg glucose uptake observed without lipid. The injected insulin was instead detected in the venous outflow from the leg. Lipid infusion caused intramuscular insulin to be diverted from interstitium into the capillary circulation, preventing a rise in intersitial insulin and any increase in local leg glucose uptake. This diversion of insulin from the interstitium under hyperlipidemic conditions may play a role in the insulin resistance observed in obesity. Further studies will be necessary to determine the mechanism for FFA’s ability to affect insulin’s hemodynamic actions.
Anti-Inflammatory Effects of Cardiovascular Exercise: Role of Visceral Adipose Tissue
Victoria Jeanne Vieira, Ph.D.
University of Illinois at Urbana–Champaign, 2009
Chronic consumption of a diet high in saturated fat (HFD) combined with a sedentary lifestyle has led to the obesity epidemic and its associated metabolic complications. At the heart of the metabolic aberrations that lead to obesity-associated diseases is inflammation that occurs in the visceral white adipose tissue (WAT). Reducing WAT inflammation, even in the absence of body weight changes, improves the metabolic consequences of obesity, highlighting the importance of the physiology of the WAT in the treatment of metabolic diseases. Exercise (EX) has been shown to reduce systemic inflammation, and this effect may be mediated by an EX-associated reduction in WAT inflammation. The overarching hypothesis of this work was that EX lowers inflammation in the visceral WAT as well as in the periphery, and that the mechanism involves, but is not fully explained by, a reduction in visceral WAT. This hypothesis was tested using 1 human intervention trial and 2 animal studies. In the first study, previously sedentary older adults participated in 10 months of cardiovascular (Cardio) or non-cardiovascular (Flex) EX. Cardio experienced significant improvements in fitness, systemic inflammation (as measured by serum C-reactive protein, CRP), as well as total and central (ie, trunk) fat. Only the decrease in trunk fat was significantly related to the reduction in CRP, suggesting that the mechanism behind the anti-inflammatory effect of EX may involve a reduction in visceral WAT. In the second study, Balb/c mice were fed an HFD for 12 weeks and then were randomized to 1 of 4 groups where they either remained on HFD and sedentary (HFSED), were exercise trained (HFEX), switched to an LFD (LFSED), or switched to an LFD and exercise trained (HFEX) for 12 weeks. LFD and EX had differential effects on WAT gene transcription (MCP-1, F4/80, IL1ra), IR, and HS. In the final animal study, C57BL/6 mice were fed an HFD for 6 weeks and then were randomized to HFSED, HFEX, LFSED, or LFEX for a 6- or a 12-week intervention. EX and LFD both decreased weight gain and relative body fat, although LFD had a more robust effect than EX. Reductions in visceral WAT explained the decreases in WAT inflammation, IR, and HS seen at 6 weeks. However, by 12 weeks, unique independent effects of EX and LFD emerged such that both treatments reduced WAT inflammation and metabolic complications. WAT macrophage infiltration was the most important independent predictor of IR whereas visceral fat mass most strongly predicted HS. In summary, there are unique metabolic consequences of a sedentary lifestyle and chronic consumption of an HFD. Both LFD and EX are critically important behavioral strategies to improve WAT health and whole body metabolic function.
Insulin Signaling and Function in Osteoblasts
Keertik S. Fulzele, Ph.D.
University of Alabama at Birmingham, 2009
Insulin and insulin-like growth factor-1 (IGF-1) are evolutionarily conserved hormonal signaling pathways with structurally similar ligands and receptors. Recent studies suggest that insulin and IGF-1 exert distinct as well as overlapping functions to regulate different aspects of skeletal development. A major problem in distinguishing the actions of insulin and IGF-1 is the fact that the receptors are co-expressed in many cell types and each ligand is able to cross-activate the other ligands’ receptor. To distinguish direct skeletal actions of insulin from that of IGF-1, we have conditionally disrupted each receptor in vitro and in vivo specifically in osteoblasts. Studies using osteoblasts lacking the IGF-1R in vitro have allowed us to demonstrate that insulin exerts direct anabolic actions in osteoblasts by activation of its cognate receptor, and that the strength of insulin generated signals is tempered through interactions with IGF-1R. Moreover, insulin treatment of DIGF-1R osteoblasts rescues the differentiation defect in these cells whereas the differentiation defect in DIR osteoblasts cannot be rescued by IGF-1 treatment.
To unequivocally establish insulin actions in bone, we have compared the phenotypes of mice that lack either the insulin receptor (Ob-ΔIR) or the IGF-1 receptor (Ob-ΔIGF-1R) in osteoblasts using a Cre/loxP recombination technique. Mice lacking the IR in osteoblasts failed to accumulate bone primarily due to a reduction in number and/or activity of osteoblasts. In contrast, mice lacking IGF-1R in osteoblasts also had reduced trabecular bone but exhibited normal or even elevated numbers of osteoblasts. The primary defect in these mice was due to a failure to mineralize osteoid matrix. As Ob-ΔIR mice aged, they developed features resembling those seen in metabolic syndrome including increased peripheral fat, glucose intolerance, and insulin insensitivity. These changes were accompanied by decreased serum adiponectin. Most importantly, circulating undercarboxylated osteocalcin, a recently identified secretagogue for insulin, was decreased in serum from Ob-ΔIR mice. Our findings indicate that insulin signaling regulates postnatal bone acquisition through mechanisms distinct from IGF-1. Moreover, insulin action in osteoblasts also influences fat accumulation, likely by regulating secretion and bioavailability of osteocalcin.
Interactive Effects of Macronutrients on Obesity and Metabolic Syndrome Development
Barbara Elise Yudell, Ph.D.
University of Illinois at Urbana–Champaign, 2009
Diet could play a key role in intervention of the obesity epidemic. However, it is unclear to what extent macronutrient composition contributes to obesity development. Previously, obesity and metabolic syndrome in rodents has been induced by either high-fat, marginally protein-deficient, or high-fructose diets. The current research investigated the interactive effects of these dietary treatments.
In the first study, male weanling Sprague-Dawley rats (N = 36) were fed 1 of 6 diets for 8 weeks. Diets were based on a 3 × 2 factorial with 3 levels of protein (P10, P15, and P25, percent energy) and 2 levels of fat (F10 and F70, percent energy). Carbohydrate (C) varied depending on protein and fat. Body composition was measured biweekly. Initially, P10 and P15 groups were hyperphagic compared to P25 groups in an attempt to meet protein requirements. However, over 8 weeks, this compensation declined, and energy intake among all groups became nearly equal. Lean body mass growth was slightly delayed in P10 groups compared to P15 and P25 groups, but all groups followed similar patterns over time. Fat mass and percent body fat were differentially affected by diet over time. In P10 groups, percent fat was relatively constant over 8 weeks, while in P25 groups, percent fat increased at each time point (P < 0.001). As expected, in P25 groups, high dietary fat caused significantly higher fat mass and percent fat (P < 0.01). In P15, this trend was also observed, although not significant. However, in P10 groups, this trend was flipped, and high dietary fat instead caused lower fat mass and percent body fat (P = 0.09). P10 groups had higher protein efficiency, as expected (P < 0.001), but unexpectedly, F70 also enhanced protein efficiency (P < 0.05). There was no difference in expression of fatty acid synthase (FAS) mRNA between P10-F10-C80 (highest percent fat) and P25-F10-C65 (lowest percent fat) in liver or adipose at 8 weeks. However, tribbles homolog 3 (TRB3) was decreased in F70 groups (P < 0.001), suggesting stimulation of lipid storage. In muscle, peroxisome proliferator-activated receptor d (PPARd), PPARg coactivator-1a (PGC-1a), and fatty acid transport protein 1 (FATP1) expression was down-regulated in F70 groups, suggesting muscle insulin resistance due to intracellular lipid accumulation.
In the second study, male Sprague-Dawley rats (N = 24) were fed 1 of 6 diets for 30 days. Diets were based on a 2 × 3 factorial with 2 levels of protein (P10 and P20, percent energy), and 3 levels of fructose (F0, F20, and F40, percent energy, replacing glucose). Body composition was measured at the beginning and end points. P10 groups showed higher energy intake (P < 0.0001) and percent fat (P < 0.01) compared to P25 groups. Unexpectedly, replacing glucose with fructose attenuated this low-protein effect without reducing lean mass. P10-40 had highest protein efficiency overall, which was 14% higher than P10-F0 (P < 0.05). While fructose tended to increase blood pressure (P = 0.06), this fructose effect was independent of adiposity. In liver, the integrated stress response (ISR) pathway was induced by low protein and fructose, suggesting inhibition of insulin signaling. However, glycogen was increased by low protein in both liver and muscle with concomitant decrease in protein tyrosine phosphatase 1B (PTP1B) expression, suggesting partial up-regulation of insulin signaling. In muscle, the glucose transporter GLUT4 and PGC-1a were induced by both low protein and fructose. Low protein also induced pyruvate dehydrogenase 4 (PDK4) in skeletal muscle, suggesting decreased glucose oxidation but not glycolysis. Low protein increased plasma adiponectin (P < 0.05), suggesting an enhanced role of adipose in energy metabolism. Increased protein efficiency by fructose may be mediated by induction of ISR in liver, and by GLUT4 and PGC-1a in muscle. Interactions of protein with carbohydrate metabolism support a significant contribution of protein to glycemic control when dietary protein is adequate. In total, these studies have confirmed the role of marginal protein deficiency in driving hyperphagia and increased adiposity, but have refuted other studies showing increased adiposity by dietary fat and fructose. When protein is marginally deficient, replacing glucose with fat or fructose unexpectedly spared amino acids and enhanced protein efficiency. No single macronutrient combination gave rise to metabolic syndrome, but instead, each diet was associated with unique metabolic regulation.
Central Artery Stiffness in Individuals With Metabolic Syndrome: Lifestyle Modification and Its Long-Term Effect on Carotid Artery Stiffness
Kunihiko Aizawa, Ph.D.
University of Western Ontario, Canada, 2009
Cardiovascular disease (CVD) is reaching an epidemic level. Not only are populations aging but also CVD risk factors often coexist, a condition known as the metabolic syndrome (MS) that increases CVD risk. An increase in arterial stiffness has emerged as one of the possible mechanisms that link MS and CVD. Therefore, a strategy to reduce arterial stiffness may also reduce CVD risks. The overall objective of this thesis was to examine the effectiveness of lifestyle modification prescribed by family physicians on central artery stiffness in individuals with MS. The first study (Chapter 2) revealed that the presence of MS increased carotid artery stiffness in middle-aged and older individuals (mean of 53.5 years) but the increase was not synergistic. The finding led to the second study (Chapter 3) determining whether a 24-week lifestyle modification would reduce carotid artery stiffness in individuals with MS. The results demonstrated that the 24-week lifestyle modification program prescribed by family physicians effectively reduced carotid artery stiffness as well as some components of MS (blood pressure, waist circumference, and blood glucose). Due to the relationship between arterial stiffness and endothelial function, the subsequent study (Chapter 4) investigated the effect of the same lifestyle modification used in the previous study on endothelial function. Despite improvements in blood pressure, waist circumference, and glucose, the intervention had no impact on endothelial function. The final study (Chapter 5) investigated how carotid artery stiffness would change with a 1-year lifestyle modification, a long period of washout (mean of 26.9 months) from the active lifestyle intervention, and a subsequent 24-week angiotensin receptor blocker telmisartan treatment in individuals at risk for CVD. Interestingly, the reduced carotid artery stiffness achieved with the 1-year lifestyle intervention was maintained following the cessation of active lifestyle intervention. Overall, these results suggest that lifestyle modification prescribed by family physicians can effectively reduce carotid artery stiffness in individuals at risk for CVD such as MS. They also suggest that the involvement of family physicians may have a sustainable effect on carotid artery stiffness even after the cessation of active lifestyle intervention by regular interaction with their patients.
Targeted Perfluorocarbon Nanoparticles for Molecular Imaging of Cardiovascular Disease and Cancer With MRI
Kejia Cai, Ph.D.
Washington University in St. Louis, Missouri, 2009
Targeted perfluorocarbon (PFC) nanoparticles are well suited for many molecular imaging applications due to their ability to carry very large payloads of imaging agents. We have developed the first targeted PARACEST (PARAmagnetic Chemical Exchange Saturation Transfer) PFC nanoparticles, which can provide “contrast on demand.” Utilizing a fibrin targeting ligand, the nanoparticles delivered a large payload of PARACEST chelates to the surface of thrombi, yielding a contrast to noise ratio of >10. In addition, the PFC core (19 F) enables quantitation of particle binding with high signal intensity and no background signal. In addition to molecular imaging applications, PARACEST PFC nanoparticles can be used for cell labeling and tracking based on their 2 unique signatures, PARACEST contrast and 19 F signal. Labeling of melanoma cells provided a PARACEST contrast of >15% in vitro, which was co-localized with the 19 F signal arising from the particle core.
Regarding potential clinical applications of this system, metabolic syndrome is an increasingly common chronic illness that promotes atherosclerosis. Plaque angiogenesis can serve as an early surrogate marker for the development of atherosclerosis. Molecular imaging of angiogenesis with targeted PFC nanoparticles might be useful for early identification of lesions susceptible to rupture and clinical sequela. Accordingly, angiogenesis in the abdominal aorta of a rat model of metabolic syndrome was studied with a vb3-integrin targeted paramagnetic nanoparticles. Obese untreated rats displayed significantly higher MR signal enhancement compared to age-matched lean rats or obese rats treated with the hypolipidemic drug Benfluorex. Immunohistological staining of angiogenic vessels in the abdominal aorta confirmed the elevated neovascularity in obese untreated animals. We conclude that MR molecular imaging with PFC nanoparticles provides a novel and sensitive means to detect molecular biomarkers associated with atherosclerosis and to monitor treatment responses.
A Murine Model of Developmental Programming of Atherosclerosis
Nima Goharkhay, Ph.D.
University of Texas Medical Branch, Graduate School of Biomedical Sciences, 2009
Early life is increasingly being recognized as an important period of development during which environmental changes can lead to long-term effects on an individual’s health. The association between poor nutrition prior to birth and an increased risk to develop coronary heart disease, hypertension, and the metabolic syndrome is well established. Animal models are a central tool to investigate the details and mechanistic basis of the effects of the early life milieu.
Coronary artery disease secondary to atherosclerosis remains a major cause of death in most societies. Limited human studies indicate a strong association between maternal hypercholesterolemia and increased rates of formation of atherosclerotic lesions in children. It is conceivable that exposure to a high lipid environment during intrauterine development and early postnatal life may emerge as one of the principal risk factors for premature atherosclerosis.
These studies were performed to determine the effect of maternal hypercholesterolemia on the risk of atherosclerotic lesion formation in the offspring in a homogeneous small animal model. The apoprotein E (apoE)-deficient mouse strain was chosen because of its well-described propensity to spontaneously manifest hypercholesterolemia and atherosclerosis. A strong correlation between maternal hypercholesterolemia and an increase in serum cholesterol levels was revealed in chow-fed heterozygous litters born to hyperlipidemic dams at both 4 and 8 months of age. In addition, 8-month-old heterozygote animals born to apoE-deficient mothers (apoE+/− mat) showed higher rates of atherosclerosis and evidence of liver and kidney damage as compared with their apo E+/− pat counterparts. In contrast, at day 21 of life apoE−/− KO and apoE+/− mat pups showed lower total cholesterol and triglyceride levels than apoE+/+ WT or apoE+/− pat litters.
Studies in liver tissue from offspring at 8 months of age suggest activation of the endogenous cholesterol synthetic pathway in apoE+/− mat offspring. This may be one of the mechanisms responsible for the observed programming effects. In vivo activity and blood pressure measurements and vascular reactivity experiments in 4-month-old animals did not demonstrate significant differences among study groups. No marked variation in serum cholesterol levels among genetically similar dams was detected.
The Role of Mitogen-Activated Protein Kinase Phosphatase 1 (MKP-1) in Metabolic Homeostatis
Rachel Jane Roth, Ph.D.
Yale University, 2009
Metabolic syndrome is increasing in prevalence in the western world at an alarming rate and is defined by multiple sequelae including obesity, insulin resistance, hepatic steatosis, cardiovascular disease, and hyperlipidemia. The molecular mechanisms controlling the development of metabolic syndrome remain unclear. Though it is generally accepted that mitogen-activated protein kinases (MAPKs) play important roles in metabolic processes, little is known about the function of the inactivators of MAPKs, the MAP kinase phosphatases (MKPs), in metabolism.
MKP-1 is ubiquitously expressed, localized to the nucleus, and induced by multiple stimuli. MKP-1 dephosphorylates all 3 MAPKs with a specificity of p38MAPK ≥ JNK > Erk. Though the role of MKP-1 in a cellular context has been thoroughly investigated, the role of MKP-1 in vivo has yet to be fully defined. Here, we report that MKP-1 plays a nonredundant role in regulation of MAPK activity in several different tissues in vivo. Mice lacking MKP-1 expression are resistant to diet-induced obesity and have enhanced energy expenditure.
Though resistant to weight gain, mkp-1−/− mice still succumb to glucose intolerance upon high fat feeding, an observation that challenges the idea that insulin sensitivity inversely correlates with weight. These data may be explained by the fact that MKP-1 inhibits nuclear MAPK activity, which modulates metabolic gene expression, whereas MAPK signaling in the cytosol regulates insulin signaling events.
In order to gain mechanistic insight into why mkp-1−/− mice are resistant to obesity, we investigated the role of MKP-1 in skeletal muscle and found that its expression increases upon high fat feeding, which is commensurate with a decrease in skeletal muscle oxidative capacity. Notably, HFD-fed mkp-1−/− mice are resistant to these changes. This may be explained at the molecular level as MKP-1 negatively regulates the stability of PGC-1a, a transcriptional coactivator and master regulator of metabolism. We further investigated the role of MKP-1 as a regulator of hepatic lipid metabolism, and found that MKP-1 expression is involved in the development of hepatic steatosis.
Taken together, this dissertation provides evidence that MKP-1 regulates metabolism in multiple tissues and gives insight to the mechanisms by which MKP-1 may regulate these processes.