Faculty Bibliography

PURPOSE: Selection of a balanced diet has a determinant impact on human health. Individual food preferences involve socio-cultural as well as physiological factors and evolve during aging. In mammals, physiological mechanisms governing food choices appear to require the sensing of nutrient concentrations in diet. This is particularly the case for dietary amino acids that are sensed by the protein kinase GCN2. It has been reported that GCN2 is involved in the adaptive response to amino acid imbalanced diets at the level of food intake and lipid metabolism. Here, we hypothesized that GCN2 may play a role in macronutrient selection and its age-related changes. METHODS: Two groups of wild-type and GCN2 knock-out mice were subjected to a food self-selection protocol at ages 6, 12, 18 and 24 months. During each test, mice were allowed to create their own diets by selecting between three separate food sources, each containing either protein, fat or carbohydrates. RESULTS: Our results show that the absence of GCN2 had two main age-related effects. First, it exacerbated fat preference at the expense of carbohydrate consumption. Second, it prevented the increase in protein intake. CONCLUSION: These findings indicate that, in omnivores, the GCN2 ancient pathway participates in the control of food preference.

Latent transforming growth factor beta (TGF-beta) binding proteins (LTBPs) are large extracellular glycoproteins structurally similar to fibrillins. They perform intricate and important roles in the extracellular matrix (ECM) and perturbations of their function manifest as a wide range of diseases. LTBPs are major regulators of TGF-beta bioavailability and action. In addition, LTBPs interact with other ECM proteins-from cytokines to large multi-factorial aggregates like microfibrils and elastic fibers, affecting their genesis, structure, and performance. In the present article, we review recent advancements in the field and relate the complex roles of LTBP in development and homeostasis. J. Cell. Biochem. 113: 410-418, 2012. (c) 2011 Wiley Periodicals, Inc

The Wnt and Hedgehog signaling pathways are essential for normal development and are misregulated in cancer. The casein kinase family of serine/threonine kinases regulates both pathways at multiple levels. However, it has been difficult to determine whether individual members of this family have distinct functions in vivo, due to their overlapping substrate specificities. In Drosophila melanogaster, photoreceptor differentiation is induced by Hedgehog and inhibited by Wingless, providing a sensitive system in which to identify regulators of each pathway. We used a mosaic genetic screen in the Drosophila eye to identify mutations in genes on the X chromosome required for signal transduction. We recovered mutations affecting the transcriptional regulator CREB binding protein, the small GTPase dynamin, the cytoskeletal regulator Actin-related protein 2, and the protein kinase Casein kinase 1alpha. Consistent with its reported function in the beta-Catenin degradation complex, Casein Kinase 1alpha mutant cells accumulate beta-Catenin and ectopically induce Wingless target genes. In contrast to previous studies based on RNA interference, we could not detect any effect of the same Casein Kinase 1alpha mutation on Hedgehog signaling. We thus propose that Casein kinase 1alpha is essential to allow beta-Catenin degradation and prevent inappropriate Wingless signaling, but its effects on the Hedgehog pathway are redundant with other Casein kinase 1 family members.

Background/purpose: Wound healing is a complex process that involves multiple intercellular and intracellular processes and extracellular interactions. Explanted human skin has been used as a model for the re-epithelialization phase of human wound healing. The currently used standard technique uses a circular punch biopsy tool to make the initial wound. Despite its wide use, the geometry of round wounds makes it difficult to measure them reliably. Methods: Our group has designed a linear wounding tool, and compared the variability in ex vivo human linear and circular wounds. Results: An F test for differences in variances demonstrated that the linear wounds provided a population of wound size measurements that was 50% less variable than that obtained from a group of matched circular wounds. This reduction in variability would provide substantial advantages for the linear wound technique over the circular wound punch technique, by reducing the sample sizes required for comparative studies of factors that alter healing. Conclusion: This linear wounding tool thus provides a method for wounding that is standardized, provides minimal error in wound gap measurements, and is easily reproducible. We demonstrate its utility in an ex vivo model for the controlled investigation of human skin wounds

We present a major revision of the iterative helical real-space refinement (IHRSR) procedure and its implementation in the SPARX single particle image processing environment. We built on over a decade of experience with IHRSR helical structure determination and we took advantage of the flexible SPARX infrastructure to arrive at an implementation that offers ease of use, flexibility in designing helical structure determination strategy, and high computational efficiency. We introduced the 3D projection matching code which now is able to work with non-cubic volumes, the geometry better suited for long helical filaments, we enhanced procedures for establishing helical symmetry parameters, and we parallelized the code using distributed memory paradigm. Additional features include a graphical user interface that facilitates entering and editing of parameters controlling the structure determination strategy of the program. In addition, we present a novel approach to detect and evaluate structural heterogeneity due to conformer mixtures that takes advantage of helical structure redundancy.

BACKGROUND: -Progressive familial heart block type I (PFHBI) is a hereditary arrhythmia characterized by progressive conduction disturbances in the His-Purkinje system. PFHBI has been linked to genes such as SCN5A that influence cardiac excitability, but not to genes that influence cell-to-cell communication. Our goal was to explore whether nucleotide substitutions in genes coding for connexin proteins would associate with clinical cases of PFHBI and if so, to establish a genotype-cell phenotype correlation for that mutation. METHODS AND RESULTS: -We screened 156 probands afflicted with PFHBI. In addition to 12 sodium channel mutations, we found a germline GJA5 (connexin40; Cx40) mutation (Q58L) in an afflicted family. Heterologous expression of Cx40-Q58L in connexin-deficient neuroblastoma cells resulted in marked reduction of junctional conductance (Cx40-WT: 22.2+/-1.7 nS, n=14; Cx40-Q58L: 0.56+/-0.34 nS, n=14; p<0.001) and diffuse localization of immunoreactive proteins in the vicinity of the plasma membrane without formation of gap junctions. Heteromeric co-transfection of Cx40-WT and Cx40-Q58L resulted in homogenous distribution of proteins in the plasma membrane rather than in membrane plaques in about 50% of cells; well-defined gap junctions were observed in other cells. Junctional conductance values correlated with the distribution of gap junction plaques. CONCLUSIONS: -Mutation Cx40-Q58L impairs gap junction formation at cell-cell interfaces. This is the first demonstration of a germline mutation in a connexin gene that associates with inherited ventricular arrhythmias, and emphasizes the importance of Cx40 in normal propagation in the specialized conduction system

Introduction Atherosclerosis is characterized by the accumulation of low density lipoprotein (LDL) and recruited macrophages within arterial walls. High macrophage burden is an indicator of greater risk of atherosclerotic tissue rupture and heart attack. As computed tomography (CT) imaging is the best technique for imaging plaque in the coronary arteries, a CT contrast agent able to detect macrophages in the arteries could help identify patients at higher risk. This study investigated whether in vivo macrophage imaging using clinical scanners could be performed with gold core high density lipoprotein (Au-HDL), a macrophage targeted agent (A). Additionally, lowest effective dose and ideal imaging timeframe of AuHDL was probed. Methods & Results Dodecanethiol coated gold cores were prepared following Brust's method and subsequently coated with the phospholipid myristoyl hydroxy phosphocholine (MHPC). The nanoparticles were then purified through centrifugation to remove gold core aggregates and empty MHPC micelles. Negative stain transmission electron microscopy (TEM) images verified removal of empty MHPC micelles from solution after ultracentrifugation (B). Apolipoprotein AI (ApoAI) was added to form the final AuHDL nanoparticle. CT imaging was used to calculate gold concentrations of the samples in mg/ml. To induce atherosclerosis, male New Zealand white rabbits were fed a high fat, high cholesterol diet (4.7% coconut oil and 0.3% cholesterol enriched diet) and underwent a double balloon injury of the aorta. AuHDL was prepared such that five atherosclerotic rabbits were injected with 75 (n=2), 150 (n=2), or 300 (n=1) mg Au/kg. CT images of rabbit aortas were taken at the following three time points: pre-injection, 24 hours post-injection, and 48 hours post-injection. A custom made MATLAB program was created to measure various regions of and around the aorta. CT images of aorta walls after injection exhibited greater radiodensity compared to pre-injection images. For example, CT images taken of one rabbit injected with 150 mg Au/kg showed the radiodensity of the aorta on average to be 38 HU (Hounsfield Units) +/- 1.99 preinjection and 59 HU +/- 1.59 24 hours postinjection (C). The lowest effective dose tested was 75 mg Au/kg and best imaging timeframe tested was 24 hours postinjection. TEM images of rabbit aorta sections confirmed localization of AuHDL nanoparticles in macrophages (D). Conclusion AuHDL increased radiodensity in CT images of aortas 24 and 48 hours post-injection compared to pre-injection images. Electron microscopy showed the nanoparticles to target macrophages. Hence this agent can image macrophages using CT, and has the potential for doing so in patients. Clinical Relevance If translated clinically, AuHDL can be used to image plaques in human aortas with high macrophage burden, thus allowing identification of patients at high risk of a heart attack. In addition, the agent could be of use in studying atherosclerosis and the effect of interventions. (Figure presented)

Rationale: Inflammation drives progression and destabilization of atherosclerotic plaques. Statins constitute the backbone for strategies to lower cardiovascular risk because of their potent cholesterol lowering capability. Whereas preclinical studies have shown that statins also have anti-inflammatory effects, the clinical relevance is hampered by the limited bioavailability of orally administered statins. To enhance the anti-inflammatory effects we developed statin-loaded reconstituted high-density lipoprotein nanoparticle ([s]-rHDL). The advantages of [s]-rHDL comprise its long half-life in plasma and the targeting to macrophages in atherosclerotic plaques. Methods & Results: To focus on anti-inflammatory effects, we used ApoE KO mice, whose cholesterol level is unaffected by statins. First, to evaluate macrophage targeting by [s]-rHDL, Gd-DTPA labeled [s]-rHDL was administered intravenously to ApoE KO mice (n=3). In vivo T1-weighted MR imaging (9.4 T Bruker MRI scanner) revealed strong signal enhancement in the abdominal aortic wall (Suppl. Fig a-d). Moreover, accumulation of [s]-rHDL was observed in the aortic valve and branching areas in the mice administered with Cy5.5 labeled [s]-rHDL by NIRF imaging (Suppl. Fig e, f) and specific uptake of [s]-rHDL by macrophages was revealed by fluorescence microscopy (Suppl. Fig g-l). Furthermore, flow cytometry confirmed that macrophages robustly took up rHDL in plaques, and the more differentiated macrophages took up more rHDL than less differentiated macrophages (Suppl. Fig m-r). Second, to assess the anti-inflammatory effects of [s]-rHDL, mice (n=62) were put on a high fat diet from 4 weeks of age onwards. At 14 weeks after diet initiation, mice were randomized to receive either placebo (n=15), oral simvastatin (10 mg/kg per day; n=15), intravenous rHDL (10 mg/kg ApoAI twice per week; n=16), or intravenous [s]-rHDL (15 mg/kg simvastatin with 10 mg/kg ApoAI twice a week; n=16) for 12 weeks. In vivo MR imaging of abdominal aorta was performed in 8 mice of each group at baseline, 6, and 12 weeks after randomization. Progression of vessel wall thickness was significantly inhibited in [s]-rHDL-treated animals compared to oral simvastatin, rHDL, and placebo groups (panel a). To objectively and quantitatively analyze histological sections (n4000), we built an automated Matlab procedure. Histology results at termination showed that plaque size (hematoxylin phloxine saffron staining) in the [s]-rHDL treated group was significantly reduced compared to rHDL and placebo groups (panel b). Importantly, the macrophage positive area (anti-CD68 immunostaining) in the [s]- rHDL treated group was profoundly reduced compared to all other groups (panel c). Conclusion: [s]-rHDL successfully delivers simvastatin to macrophages in atherosclerotic plaques as revealed by in vivo MRI imaging, ex vivo imaging, and histology. As a consequence, the [s]-rHDL formulation improves the anti-inflammatory effects of statins, which can be expected to improve its atheroprotective effects compared to oral statin therapy. These data warrant further studies in patients at increased cardiovascular risk. (Figure presented)

AIMS/HYPOTHESIS: Insulin activates insulin receptor protein tyrosine kinase and downstream phosphatidylinositol-3-kinase (PI3K)/Akt signalling in muscle to promote glucose uptake. The insulin receptor can serve as a substrate for the protein tyrosine phosphatase (PTP) 1B and T cell protein tyrosine phosphatase (TCPTP), which share a striking 74% sequence identity in their catalytic domains. PTP1B is a validated therapeutic target for the alleviation of insulin resistance in type 2 diabetes. PTP1B dephosphorylates the insulin receptor in liver and muscle to regulate glucose homeostasis, whereas TCPTP regulates insulin receptor signalling and gluconeogenesis in the liver. In this study we assessed for the first time the role of TCPTP in the regulation of insulin receptor signalling in muscle. METHODS: We generated muscle-specific TCPTP-deficient (Mck-Cre;Ptpn2(lox/lox)) mice (Mck, also known as Ckm) and assessed the impact on glucose homeostasis and muscle insulin receptor signalling in chow-fed versus high-fat-fed mice. RESULTS: Blood glucose and insulin levels, insulin and glucose tolerance, and insulin-induced muscle insulin receptor activation and downstream PI3K/Akt signalling remained unaltered in chow-fed Mck-Cre;Ptpn2(lox/lox) versus Ptpn2(lox/lox) mice. In addition, body weight, adiposity, energy expenditure, insulin sensitivity and glucose homeostasis were not altered in high-fat-fed Mck-Cre;Ptpn2(lox/lox) versus Ptpn2(lox/lox) mice. CONCLUSIONS/INTERPRETATION: These results indicate that TCPTP deficiency in muscle has no effect on insulin signalling and glucose homeostasis, and does not prevent high-fat diet-induced insulin resistance. Thus, despite their high degree of sequence identity, PTP1B and TCPTP contribute differentially to insulin receptor regulation in muscle. Our results are consistent with the notion that these two highly related PTPs make distinct contributions to insulin receptor regulation in different tissues.

ER-associated degradation (ERAD) rids the early secretory pathway of misfolded or misprocessed proteins. Some members of the protein disulfide isomerase (PDI) family appear to facilitate ERAD substrate selection and retrotranslocation, but a thorough characterization of PDIs during the degradation of diverse substrates has not been undertaken, in part because there are 20 PDI family members in mammals. PDIs can also exhibit disulfide redox, isomerization, and/or chaperone activity, but which of these activities is required for the ERAD of different substrate classes is unknown. We therefore examined the fates of unique substrates in yeast, which expresses five PDIs. Through the use of a yeast expression system for apolipoprotein B (ApoB), which is disulfide rich, we discovered that Pdi1 interacts with ApoB and facilitates degradation through its chaperone activity. In contrast, Pdi1's redox activity was required for the ERAD of CPY* (a misfolded version of carboxypeptidase Y that has five disulfide bonds). The ERAD of another substrate, the alpha subunit of the epithelial sodium channel, was Pdi1 independent. Distinct effects of mammalian PDI homologues on ApoB degradation were then observed in hepatic cells. These data indicate that PDIs contribute to the ERAD of proteins through different mechanisms and that PDI diversity is critical to recognize the spectrum of potential ERAD substrates.