bluuebunny/crossref_metadata_2025 huggingface dataset:
Responses of Suprachiasmatic Nucleus Neurons to Light and Dark Adaptation: Relative Contributions of Melanopsin and Rod–Cone Inputs
Authors: Drouyer Elise , Rieux Camille , Hut Roelof A. , Cooper Howard M.
Published: September 2007

The circadian oscillator in the suprachiasmatic nucleus (SCN) is entrained to the environmental light/dark cycle through photic information conveyed from the retina. The vast majority of projections to the SCN arise from melanopsin-expressing ganglion cells that are intrinsically light sensitive and that receive inputs from both rods and cones. To investigate the relative contributions of the different photoreceptive systems in shaping the photic signal influencing the circadian clock, we analyzed neuronal responses of single SCN neurons using extracellular electrophysiological recordings under different conditions of light adaptation. In the majority of neurons (78%), the spike rate is increased by light stimulation whereas the remainder are light-inhibited. The neuronal response to light is composed of several components distinguished by their temporal dynamics and degree of alteration after previous light exposure. SCN neurons display a sustained response to light followed by persistence of the response after light offset. These responses are sluggish and relatively unaffected by previous light exposures. Neurons also respond with a brisk, excitatory ON response and often an OFF response that is either excitatory or inhibitory. ON–OFF responses are transient and strongly reduced by previous bright white light exposure. Furthermore, two types of neuronal response patterns can be distinguished by the presence or absence of a slow-transient component that follows the transient ON response. The transient ON–OFF components express light adaptation properties characteristic of retinal channels involving cones, whereas the sustained and persistent components are consistent within vitroresponse properties reported for melanopsin-expressing ganglion cells.
Skin-Derived Precursors Generate Myelinating Schwann Cells That Promote Remyelination and Functional Recovery after Contusion Spinal Cord Injury
Authors: Biernaskie Jeff , Sparling Joseph S. , Liu Jie , Shannon Casey P. , Plemel Jason R. , Xie Yuanyun , Miller Freda D. , Tetzlaff Wolfram
Published: September 2007

Transplantation of exogenous cells is one approach to spinal cord repair that could potentially enhance the growth and myelination of endogenous axons. Here, we asked whether skin-derived precursors (SKPs), a neural crest-like precursor that can be isolated and expanded from mammalian skin, could be used to repair the injured rat spinal cord. To ask this question, we isolated and expanded genetically tagged murine SKPs and either transplanted them directly into the contused rat spinal cord or differentiated them into Schwann cells (SCs), and performed similar transplantations with the isolated, expanded SKP-derived SCs. Neuroanatomical analysis of these transplants 12 weeks after transplantation revealed that both cell types survived well within the injured spinal cord, reduced the size of the contusion cavity, myelinated endogenous host axons, and recruited endogenous SCs into the injured cord. However, SKP-derived SCs also provided a bridge across the lesion site, increased the size of the spared tissue rim, myelinated spared axons within the tissue rim, reduced reactive gliosis, and provided an environment that was highly conducive to axonal growth. Importantly, SKP-derived SCs provided enhanced locomotor recovery relative to both SKPs and forebrain subventricular zone neurospheres, and had no impact on mechanical or heat sensitivity thresholds. Thus, SKP-derived SCs provide an accessible, potentially autologous source of cells for transplantation into and treatment of the injured spinal cord.
Nonpsychoactive Cannabidiol Prevents Prion Accumulation and Protects Neurons against Prion Toxicity
Authors: Dirikoc Sevda , Priola Suzette A. , Marella Mathieu , Zsürger Nicole , Chabry Joëlle
Published: September 2007

Prion diseases are transmissible neurodegenerative disorders characterized by the accumulation in the CNS of the protease-resistant prion protein (PrPres), a structurally misfolded isoform of its physiological counterpart PrPsen. Both neuropathogenesis and prion infectivity are related to PrPres formation. Here, we report that the nonpsychoactive cannabis constituent cannabidiol (CBD) inhibited PrPres accumulation in both mouse and sheep scrapie-infected cells, whereas other structurally related cannabinoid analogs were either weak inhibitors or noninhibitory. Moreover, after intraperitoneal infection with murine scrapie, peripheral injection of CBD limited cerebral accumulation of PrPres and significantly increased the survival time of infected mice. Mechanistically, CBD did not appear to inhibit PrPres accumulation via direct interactions with PrP, destabilization of PrPres aggregates, or alteration of the expression level or subcellular localization of PrPsen. However, CBD did inhibit the neurotoxic effects of PrPres and affected PrPres-induced microglial cell migration in a concentration-dependent manner. Our results suggest that CBD may protect neurons against the multiple molecular and cellular factors involved in the different steps of the neurodegenerative process, which takes place during prion infection. When combined with its ability to target the brain and its lack of toxic side effects, CBD may represent a promising new anti-prion drug.
Hippocampal CA1 Place Cells Encode Intended Destination on a Maze with Multiple Choice Points
Authors: Ainge James A. , Tamosiunaite Minija , Woergoetter Florentin , Dudchenko Paul A.
Published: September 2007

The hippocampus encodes both spatial and nonspatial aspects of a rat's ongoing behavior at the single-cell level. In this study, we examined the encoding of intended destination by hippocampal (CA1) place cells during performance of a serial reversal task on a double Y-maze. On the maze, rats had to make two choices to access one of four possible goal locations, two of which contained reward. Reward locations were kept constant within blocks of 10 trials but changed between blocks, and the session of each day comprised three or more trial blocks. A disproportionate number of place fields were observed in the start box and beginning stem of the maze, relative to other locations on the maze. Forty-six percent of these place fields had different firing rates on journeys to different goal boxes. Another group of cells had place fields before the second choice point, and, of these, 44% differentiated between journeys to specific goal boxes. In a second experiment, we observed that rats with hippocampal damage made significantly more errors than control rats on the Y-maze when reward locations were reversed. Together, these results suggest that, at the start of the maze, the hippocampus encodes both current location and the intended destination of the rat, and this encoding is necessary for the flexible response to changes in reinforcement contingencies.
State Changes Rapidly Modulate Cortical Neuronal Responsiveness
Authors: Hasenstaub Andrea , Sachdev Robert N. S. , McCormick David A.
Published: September 2007

The responsiveness of cortical neurons is strongly and rapidly influenced by changes in the level of local network activity. In rodent somatosensory cortex, increases in network activity increase neuronal responsiveness to the intracellular injection of brief conductance stimuli but paradoxically decrease responsiveness to brief whisker deflections. However, whisker stimulation frequently evokes long-lasting changes in the level of local circuit activity. The ability of stimuli to successfully evoke prolonged increases in circuit activity is associated with both an increase in the amount of conductance evoked by a whisker stimulus and an increase in action potential responsiveness to whisker stimulation. In addition, brief whisker stimuli presented during periods of high network activity evoke postsynaptic potentials containing a greater proportion of inhibition, consistent with an increased efficiency in the activation of inhibitory mechanisms during the Up state. In contrast, during prolonged and variable whisker stimulation, increased network activity is associated with an increase in overall responsiveness, dynamic range, output gain, and correlation between action potential response and speed of whisker movement. We conclude that stimulus-evoked or spontaneous alterations in cortical state can influence neuronal responsiveness in a complex manner, resulting in large changes in which, and how, sensory stimuli are represented.
Fate-Mapping the Mammalian Hindbrain: Segmental Origins of Vestibular Projection Neurons Assessed Using Rhombomere-SpecificHoxa2Enhancer Elements in the Mouse Embryo
Authors: Pasqualetti Massimo , Díaz Carmen , Renaud Jean-Sébastien , Rijli Filippo M. , Glover Joel C.
Published: September 2007

As a step toward generating a fate map of identified neuron populations in the mammalian hindbrain, we assessed the contributions of individual rhombomeres to the vestibular nuclear complex, a major sensorimotor area that spans the entire rhombencephalon. Transgenic mice harboring either thelacZor the enhanced green fluorescent protein reporter genes under the transcriptional control of rhombomere-specificHoxa2enhancer elements were used to visualize rhombomere-derived domains. We labeled functionally identifiable vestibular projection neuron groups retrogradely with conjugated dextran-amines at successive embryonic stages and obtained developmental fate maps through direct comparison with the rhombomere-derived domains in the same embryos. The fate maps show that each vestibular neuron group derives from a unique rostrocaudal domain that is relatively stable developmentally, suggesting that anteroposterior migration is not a major contributor to the rostrocaudal patterning of the vestibular system. Most of the groups are multisegmental in origin, and each rhombomere is fated to give rise to two or more vestibular projection neuron types, in a complex pattern that is not segmentally iterated. Comparison with studies in the chicken embryo shows that the rostrocaudal patterning of identified vestibular projection neuron groups is generally well conserved between avians and mammalians but that significant species-specific differences exist in the rostrocaudal limits of particular groups. This mammalian hindbrain fate map can be used as the basis for targeting genetic manipulation to specific subpopulations of vestibular projection neurons.
Estrogen Disrupts the Inhibition of Fear in Female Rats, Possibly through the Antagonistic Effects of Estrogen Receptor α (ERα) and ERβ
Authors: Toufexis Donna J. , Myers Karyn M. , Bowser Michael E. , Davis Michael
Published: September 2007

The ambiguous role of estrogen in emotional learning may result from opposing actions of estrogen receptor α (ERα) and ERβ. Using a fear-conditioning paradigm called the AX+, BX− discrimination, in which cue A comes to elicit fear and cue B becomes a safety signal, we examined the effect of 17β-estradiol (E) and selective ERα and ERβ agonists on excitatory and inhibitory fear learning. Gonadectomized (GDX) male and female rats implanted with E or selective ERα or ERβ agonists were trained on the AX+, BX− discrimination and tested periodically to A, B, and AB. GDX sham-implanted male and female rats and GDX E-implanted males, but not GDX E-implanted females, exhibited less fear to AB than to A, suggesting that estrogen interferes with generalization of safety signals in female rats. ERα and ERβ agonists disrupted discrimination learning in both sexes. ERα-implanted groups had higher fear responses to all cues than did ERβ-implanted groups, suggesting that these two receptors have opposing effects in aversive discrimination learning. In contrast, neither E nor ERα and ERβ agonists affected single-cue fear conditioning in either sex. These data suggest that E does not enhance fear in emotional learning but acts to disrupt the inhibition of fear in females only.
Human Adult Cortical Reorganization and Consequent Visual Distortion
Authors: Dilks Daniel D. , Serences John T. , Rosenau Benjamin J. , Yantis Steven , McCloskey Michael
Published: September 2007

Neural and behavioral evidence for cortical reorganization in the adult somatosensory system after loss of sensory input (e.g., amputation) has been well documented. In contrast, evidence for reorganization in the adult visual system is far less clear: neural evidence is the subject of controversy, behavioral evidence is sparse, and studies combining neural and behavioral evidence have not previously been reported. Here, we report converging behavioral and neuroimaging evidence from a stroke patient (B.L.) in support of cortical reorganization in the adult human visual system. B.L.'s stroke spared the primary visual cortex (V1), but destroyed fibers that normally provide input to V1 from the upper left visual field (LVF). As a consequence, B.L. is blind in the upper LVF, and exhibits distorted perception in the lower LVF: stimuli appear vertically elongated, toward and into the blind upper LVF. For example, a square presented in the lower LVF is perceived as a rectangle extending upward. We hypothesized that the perceptual distortion was a consequence of cortical reorganization in V1. Extensive behavioral testing supported our hypothesis, and functional magnetic resonance imaging (fMRI) confirmed V1 reorganization. Together, the behavioral and fMRI data show that loss of input to V1 after a stroke leads to cortical reorganization in the adult human visual system, and provide the first evidence that reorganization of the adult visual system affects visual perception. These findings contribute to our understanding of the human adult brain's capacity to change and has implications for topics ranging from learning to recovery from brain damage.
Sequential Development of Long-Term Potentiation and Depression in Different Layers of the Mouse Visual Cortex
Authors: Jiang Bin , Treviño Mario , Kirkwood Alfredo
Published: September 2007

Visual deprivation affects the responses of layer IV cells more prominently during early postnatal development, whereas responses in layer II/III remain modifiable until later ages. We examined whether these laminar differences correlate with changes in long-term potentiation (LTP) and long-term depression (LTD) of the ascending pathways to layers IV and II/III in the mouse visual cortex. Our analysis revealed that LTP and LTD in layer IV principal cells is lost shortly after the eyes open, but persists in layers II/III beyond puberty. These results suggest that plasticity proceeds sequentially through cortical layers in a manner that parallels the flow of information during sensory processing.
Motor Neuron Disease-Associated Mutant Vesicle-Associated Membrane Protein-Associated Protein (VAP) B Recruits Wild-Type VAPs into Endoplasmic Reticulum-Derived Tubular Aggregates
Authors: Teuling Eva , Ahmed Suaad , Haasdijk Elize , Demmers Jeroen , Steinmetz Michel O. , Akhmanova Anna , Jaarsma Dick , Hoogenraad Casper C.
Published: September 2007

The vesicle-associated membrane protein-associated proteins (VAPs) VAPA and VAPB interact with lipid-binding proteins carrying a short motif containing two phenylalanines in an acidic tract (FFAT motif) and targets them to the cytosolic surface of the endoplasmic reticulum (ER). A genetic mutation (P56S) in the conserved major sperm protein homology domain of VAPB has been linked to motor-neuron degeneration in affected amyotrophic lateral sclerosis (ALS) patients. We report that in the CNS, VAPB is abundant in motor neurons and that the P56S substitution causes aggregation of mutant VAPB in immobile tubular ER clusters, perturbs FFAT-motif binding, and traps endogenous VAP in mutant aggregates. Expression of mutant VAPB or reduction of VAP by short hairpin RNA in primary neurons causes Golgi dispersion and cell death. VAPA and VAPB are reduced in human ALS patients and superoxide dismutase 1 (SOD1)-ALS-transgenic mice, suggesting that VAP family proteins may be involved in the pathogenesis of sporadic and SOD1-linked ALS. Our data support a model in which reduced levels of VAP family proteins result in decreased ER anchoring of lipid-binding proteins and cause motor neuron degeneration.
Extracellular Glutamate Concentration in Hippocampal Slice
Authors: Herman Melissa A. , Jahr Craig E.
Published: September 2007

Synaptic glutamate transients resulting from vesicular exocytosis are superimposed on a low baseline concentration of glutamate in the extracellular space. Reported values of baseline glutamate concentrations range up to 4 μm. If glutamate were present tonically at low micromolar concentrations, many receptors, especially the high-affinity NMDA receptors (NMDARs), would be activated or desensitized, altering neuronal excitability. Using NMDARs expressed by CA1 pyramidal cells in acute hippocampal slices to monitor extracellular glutamate, we find that its baseline concentration is much lower, near 25 nm. In addition, superfusion of low micromolar concentrations of glutamate had no effect on neurons, indicating that glutamate transport prevents access to receptors within the slice. However, equipotent concentrations of NMDA, a nontransported agonist, depolarized neurons dramatically. We suggest that ambient concentrations of glutamatein vivoare also in the nanomolar range and are too low to cause significant receptor activation.
Cilia Proteins Control Cerebellar Morphogenesis by Promoting Expansion of the Granule Progenitor Pool
Authors: Chizhikov Victor V. , Davenport James , Zhang Qihong , Shih Evelyn Kim , Cabello Olga A. , Fuchs Jannon L. , Yoder Bradley K. , Millen Kathleen J.
Published: September 2007

Although human congenital cerebellar malformations are common, their molecular and developmental basis is still poorly understood. Recently, cilia-related gene deficiencies have been implicated in several congenital disorders that exhibit cerebellar abnormalities such as Joubert syndrome, Meckel-Gruber syndrome, Bardet-Biedl syndrome, and Orofaciodigital syndrome. The association of cilia gene mutations with these syndromes suggests that cilia may be important for cerebellar development, but the nature of cilia involvement has not been elucidated. To assess the importance of cilia-related proteins during cerebellar development, we studied the effects of CNS-specific inactivation of two mouse genes whose protein products are critical for cilia formation and maintenance,IFT88, (also known aspolarisorTg737), which encodes intraflagellar transport 88 homolog, andKif3a, which encodes kinesin family member 3a. We showed that loss of either of these genes caused severe cerebellar hypoplasia and foliation abnormalities, primarily attributable to a failure of expansion of the neonatal granule cell progenitor population. In addition, granule cell progenitor proliferation was sensitive to partial loss of IFT function in a hypomorphic mutant ofIFT88(IFT88orpk), an effect that was modified by genetic background.IFT88andKif3awere not required for the specification and differentiation of most other cerebellar cell types, including Purkinje cells. Together, our observations constitute the first demonstration that cilia proteins are essential for normal cerebellar development and suggest that granule cell proliferation defects may be central to the cerebellar pathology in human cilia-related disorders.
Developmental Defects in Neutrophils from Preterm Infants
Authors: Chandra Sharat , Haines Hillary , Michie Colin , Maheshwari Akhil
Published: September 2007

Neutrophil development starts in the early second trimester in the human fetus and continues through the rest of the gestation. Preterm birth can interrupt the maturation of fetal neutrophils and place the preterm neonate at risk of life-threatening infections. Various developmental defects have been identified in preterm neutrophils, such as in the ability of circulating neutrophils to cross the endothelial barrier and abnormalities in chemotaxis, respiratory burst, and degranulation. The availability of recombinant human myeloid growth factors has renewed interest in understanding the mechanisms and natural history of such defects. In this article, we review various aspects of the developmental immaturity of preterm neutrophils.
Magnesium and Perinatal Asphyxia
Authors: Leone Cléa R. , Barbosa Naila O.E.
Published: September 2007

The pathophysiology of perinatal hypoxic-ischemic insults has been investigated exhaustively to identify the components that must be blocked to reduce neurologic injury in the newborn. Among potential therapeutic strategies for neuroprotection, administration of magnesium has been the object of experimental studies and, recently, clinical trials. This interest is related to the compound's potential effect of blocking glutamate-controlled N-methyl-D-aspartate (NMDA) receptors and the voltage-dependent calcium channels, preventing the influx of extracellular calcium into the neurons, as well as its action as a membrane stabilizer. To date, results have been variable and are not sufficient to recommend this therapy in newborns who have perinatal asphyxia.
Embankment supported on piles with biaxial geogrids
Authors: Almeida M. S. S. , Ehrlich M. , Spotti A. P. , Marques M. E. S.
Published: September 2007

The case study consists of a piled embankment built over a very soft, organic compressible clay layer 10 m deep. The height of the embankment supported on piles was about 1·3 m, and the piles, which measured 0·18 m × 0·18 m, were spaced 2·5 m apart in a square pattern, with 0·8 m wide square pile caps covered with a bidirectional 200 kN/m geogrid polyester reinforcement. A region of the piled supported embankment was instrumented to provide a better understanding of the overall embankment-reinforcement-pile cap system. Three-dimensional and two-dimensional pile layouts were adopted. An excavation of 1 m under the geogrid was carried out to induce rapid load transference to the geogrid. The settlement in between pile caps was 0·10–0·4 m, whereas settlements in the region under similar embankment heights were much higher, thus demonstrating the efficacy of pile-supported embankments with geogrids in controlling settlements. Strains at the reinforcement were also measured, and showed values in the range 0·25–2·05%.
A case study: delayed failure of a deep cutting in lodgement till
Authors: Hughes D. , Sivakumar V. , Glynn D. , Clarke G.
Published: September 2007

Recent research on the delayed failure of cuttings in clay clearly recognises and predicts progressive delayed failure of deep cuttings. This is due to a combination of strain-softening, weathering, dissipation of negative excess pore water pressure generated at the time of excavation, and frequent occurrence of prolonged periods of wet weather. There have been several slope failures of this kind in Northern Ireland. This paper discusses a case study based on a failure of a deep cutting, excavated at a slope of 1 in 2, on the A1 near Dromore (County Down) in Northern Ireland. The cutting was in lodgement till, a stiff, heavily overconsolidated clay. The failure occurred approximately 30 years after the cutting was excavated, following a prolonged period of heavy rainfall. An analysis of the failure, together with laboratory test data on soil samples taken from the site, confirmed that by using long-term soil strength parameters the factor of safety of this slope was unity. The conclusion of the analysis is that slopes excavated in this soil should be designed (and assessed) on long-term strength parameters.
Reducing differential settlements of approach embankments
Authors: Shen S.-L. , Hong Z.-S. , Xu Y.-S.
Published: September 2007

This paper presents a case history of an approach road remediation project on a low embankment over soft Ariake clay, located in the coastal Saga Plain of Japan. After the road had been opened to traffic for two-and-a-half years, large settlements were observed, and there was significant differential settlement between piled structures and approach road embankments on soft clay. This differential settlement had a significant impact on vehicle ride quality, and necessitated a remediation programme. In the remediation project, a new method called the column approach (CA) method was adopted to support a transitional zone at the interfaces of piled structures and road embankment. With the CA method a transitional zone was designed at these interfaces to be supported by soil-cement columns of varying lengths to smooth out the settlement profile within these zones. Field monitoring was conducted following completion of remediation. The CA method was also compared with two other Japanese code-designated methods: overlaying a new pavement on the existing pavement at a certain time interval, and constructing a concrete cushion slab under the road pavement. Both practical and economic aspects are examined in the case study presented. This study shows that, when the differential settlement is over 0·3 m, overlay remediation is not economic, and the approach cushion method does not perform well. The CA method not only reduces the differential settlement effectively, but also reduces the total construction cost, although the initial investment is relatively high.
Controlling clay pore pressures for cut-and-cover tunnelling
Authors: Roberts T. O. L. , Roscoe H. , Powrie W. , Butcher D. J. E.
Published: September 2007

To facilitate construction of the Channel Tunnel Rail Link through Ashford in cut-and-cover tunnels and retained cut, it was necessary to control pore water pressures in the relatively low-permeability, laminated Weald Clay. This was achieved by means of an ejector well dewatering system. This paper describes and discusses the investigations carried out to characterise the in situ permeability of the Weald Clay, the design and performance of the ejector well system installed, and the associated soil surface settlements. The correlation between the changes in pore pressure and settlements at the site is compared with that given by Preene et al. This comparison is used to evaluate the method, and to provide some insights into the selection of appropriate parameter values of soil permeability and stiffness.
Rectangular barrettes and circular bored piles in saprolites
Authors: Lei G. H. , Ng C. W. W.
Published: September 2007

In the last two decades, many full-scale compression loading tests have been performed to verify the design parameters of the shaft resistance of rectangular barrettes and large-diameter bored piles founding in deep-seated thick saprolites in Hong Kong. In this study, comparisons are made between the results from the investigations of the behaviour of the mobilised shaft resistance with respect to the relative pile–soil movements (i.e. local displacements), the in situ soil properties (i.e. standard penetration test values), and the effective stress principle (with which the β values are estimated). Also, the behaviour of the mobilised shaft resistance for the barrettes and bored piles in Hong Kong is compared with that elsewhere.
β-Cell Mitochondria Exhibit Membrane Potential Heterogeneity That Can Be Altered by Stimulatory or Toxic Fuel Levels
Authors: Wikstrom Jakob D. , Katzman Shana M. , Mohamed Hibo , Twig Gilad , Graf Solomon A. , Heart Emma , Molina Anthony J.A. , Corkey Barbara E. , de Vargas Lina Moitoso , Danial Nika N. , Collins Sheila , Shirihai Orian S.
Published: September 2007

OBJECTIVE—β-Cell response to glucose is characterized by mitochondrial membrane potential (ΔΨ) hyperpolarization and the production of metabolites that serve as insulin secretory signals. We have previously shown that glucose-induced mitochondrial hyperpolarization accompanies the concentration-dependent increase in insulin secretion within a wide range of glucose concentrations. This observation represents the integrated response of a large number of mitochondria within each individual cell. However, it is currently unclear whether all mitochondria within a single β-cell represent a metabolically homogenous population and whether fuel or other stimuli can recruit or silence sizable subpopulations of mitochondria. This study offers insight into the different metabolic states of β-cell mitochondria.

RESULTS—We show that mitochondria display a wide heterogeneity in ΔΨ and a millivolt range that is considerably larger than the change in millivolts induced by fuel challenge. Increasing glucose concentration recruits mitochondria into higher levels of homogeneity, while an in vitro diabetes model results in increased ΔΨ heterogeneity. Exploration of the mechanism behind heterogeneity revealed that temporary changes in ΔΨ of individual mitochondria, ATP-hydrolyzing mitochondria, and uncoupling protein 2 are not significant contributors to ΔΨ heterogeneity. We identified BAD, a proapoptotic BCL-2 family member previously implicated in mitochondrial recruitment of glucokinase, as a significant factor influencing the level of heterogeneity.

CONCLUSIONS—We suggest that mitochondrial ΔΨ heterogeneity in β-cells reflects a metabolic reservoir recruited by an increased level of fuels and therefore may serve as a therapeutic target.
Endothelial Nitric Oxide Synthase (eNOS) Knockout Mice Have Defective Mitochondrial β-Oxidation
Authors: Gouill Eric Le , Jimenez Maria , Binnert Christophe , Jayet Pierre-Yves , Thalmann Sebastien , Nicod Pascal , Scherrer Urs , Vollenweider Peter
Published: September 2007

OBJECTIVE— Recent observations indicate that the delivery of nitric oxide by endothelial nitric oxide synthase (eNOS) is not only critical for metabolic homeostasis, but could also be important for mitochondrial biogenesis, a key organelle for free fatty acid (FFA) oxidation and energy production. Because mice deficient for the gene of eNOS (eNOS−/−) have increased triglycerides and FFA levels, in addition to hypertension and insulin resistance, we hypothesized that these knockout mice may have decreased energy expenditure and defective β-oxidation.

RESEARCH DESIGN AND METHODS— Several markers of mitochondrial activity were assessed in C57BL/6J wild-type or eNOS−/− mice including the energy expenditure and oxygen consumption by indirect calorimetry, in vitro β-oxidation in isolated mitochondria from skeletal muscle, and expression of genes involved in fatty acid oxidation.

RESULTS— eNOS−/− mice had markedly lower energy expenditure (−10%, P < 0.05) and oxygen consumption (−15%, P < 0.05) than control mice. This was associated with a roughly 30% decrease of the mitochondria content (P < 0.05) and, most importantly, with mitochondrial dysfunction, as evidenced by a markedly lower β-oxidation of subsarcolemmal mitochondria in skeletal muscle (−30%, P < 0.05). Finally, impaired mitochondrial β-oxidation was associated with a significant increase of the intramyocellular lipid content (30%, P < 0.05) in gastrocnemius muscle.

CONCLUSIONS— These data indicate that elevated FFA and triglyceride in eNOS−/− mice result in defective mitochondrial β-oxidation in muscle cells.
Decreased l-Type Ca2+ Current in Cardiac Myocytes of Type 1 Diabetic Akita Mice Due to Reduced Phosphatidylinositol 3-Kinase Signaling
Authors: Lu Zhongju , Jiang Ya-Ping , Xu Xin-Hua , Ballou Lisa M. , Cohen Ira S. , Lin Richard Z.
Published: September 2007

OBJECTIVE—Contraction of cardiac myocytes is initiated by Ca2+ entry through the voltage-dependent l-type Ca2+ channel (LTCC). Previous studies have shown that phosphatidylinositol (PI) 3-kinase signaling modulates LTCC function. Because PI 3-kinases are key mediators of insulin action, we investigated whether LTCC function is affected in diabetic animals due to reduced PI 3-kinase signaling.

RESEARCH DESIGN AND METHODS—We used whole-cell patch clamping and biochemical assays to compare cardiac LTCC function and PI 3-kinase signaling in insulin-deficient diabetic mice heterozygous for the Ins2Akita mutation versus nondiabetic littermates.

RESULTS—Diabetic mice had a cardiac contractility defect, reduced PI 3-kinase signaling in the heart, and decreased l-type Ca2+ current (ICa,L) density in myocytes compared with control nondiabetic littermates. The lower ICa,L density in myocytes from diabetic mice is due at least in part to reduced cell surface expression of the LTCC. ICa,L density in myocytes from diabetic mice was increased to control levels by insulin treatment or intracellular infusion of PI 3,4,5-trisphosphate [PI(3,4,5)P3]. This stimulatory effect was blocked by taxol, suggesting that PI(3,4,5)P3 stimulates microtubule-dependent trafficking of the LTCC to the cell surface. The voltage dependence of steady-state activation and inactivation of ICa,L was also shifted to more positive potentials in myocytes from diabetic versus nondiabetic animals. PI(3,4,5)P3 infusion eliminated only the difference in voltage dependence of steady-state inactivation of ICa,L.

CONCLUSIONS—Decreased PI 3-kinase signaling in myocytes from type 1 diabetic mice leads to reduced Ca2+ entry through the LTCC, which might contribute to the negative effect of diabetes on cardiac contractility.
PTEN Expression Contributes to the Regulation of Muscle Protein Degradation in Diabetes
Authors: Hu Zhaoyong , Lee In Hee , Wang Xiaonan , Sheng Hongmiao , Zhang Liping , Du Jie , Mitch William E.
Published: September 2007

OBJECTIVE—Conditions accelerating muscle proteolysis are frequently associated with defective phosphatidylinositol 3-kinase (PI3K)/Akt signaling and reduced PI3K-generated phosphatidylinositol 3,4,5-triphosphate (PIP3). We evaluated the control of muscle protein synthesis and degradation in mouse models of type 1 and 2 diabetes to determine whether defects besides PI3K/Akt activities affect muscle metabolism.

RESEARCH DESIGN AND METHODS—We evaluated the expression and activity of PTEN, the phosphatase converting PIP3 to inactive phosphatidylinositol 4,5-bisphosphate, and studied how PTEN influences muscle protein in diabetic wild-type mice and in mice with partial deficiency of PTEN+/−.

RESULTS—In acutely diabetic mice, muscle PTEN expression was decreased. It was increased by chronic diabetes or insulin resistance. In cultured C2C12 myotubes, acute suppression of PI3K activity led to decreased PTEN expression, while palmitic acid increased PTEN in myotubes in a p38-dependent fashion. To examine whether PTEN affects muscle protein turnover, we studied primary myotubes cultures from wild-type and PTEN+/− mice. The proteolysis induced by serum deprivation was suppressed in PTEN+/− cells. Moreover, the sizes of muscle fibers in PTEN+/− and wild-type mice were similar, but the increase in muscle proteolysis caused by acute diabetes was significantly suppressed by PTEN+/−. This antiproteolytic response involved higher PIP3 and p-Akt levels and a decrease in caspase-3–mediated actin cleavage and activation of the ubiquitin-proteasome system as signified by reduced induction of atrogin-1/MAFbx or MurF1 (muscle-specific RING finger protein 1).

CONCLUSIONS—Changes in PTEN expression participate in the regulation of muscle proteolytic pathways. A decrease in PTEN could be a compensatory mechanism to prevent muscle protein losses.
Deficiency of Cbl-b Gene Enhances Infiltration and Activation of Macrophages in Adipose Tissue and Causes Peripheral Insulin Resistance in Mice
Authors: Hirasaka Katsuya , Kohno Shohei , Goto Jumpei , Furochi Harumi , Mawatari Kazuaki , Harada Nagakatsu , Hosaka Toshio , Nakaya Yutaka , Ishidoh Kazumi , Obata Toshiyuki , Ebina Yousuke , Gu Hua , Takeda Shin'ichi , Kishi Kyoichi , Nikawa Takeshi
Published: September 2007

OBJECTIVE—c-Cbl plays an important role in whole-body fuel homeostasis by regulating insulin action. In the present study, we examined the role of Cbl-b, another member of the Cbl family, in insulin action.

RESEARCH DESIGN AND METHODS—C57BL/6 (Cbl-b+/+) or Cbl-b-deficient (Cbl-b−/−) mice were subjected to insulin and glucose tolerance tests and a hyperinsulinemic-euglycemic clamp test. Infiltration of macrophages into white adipose tissue (WAT) was assessed by immunohistochemistry and flow cytometry. We examined macrophage activation using co-cultures of 3T3-L1 adipocytes and peritoneal macrophages.

RESULTS—Elderly Cbl-b−/− mice developed glucose intolerance and peripheral insulin resistance; serum insulin concentrations after a glucose challenge were always higher in elderly Cbl-b−/− mice than age-matched Cbl-b+/+ mice. Deficiency of the Cbl-b gene significantly decreased the uptake of 2-deoxyglucose into WAT and glucose infusion rate, whereas fatty liver was apparent in elderly Cbl-b−/− mice. Cbl-b deficiency was associated with infiltration of macrophages into the WAT and expression of cytokines, such as tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein (MCP)-1. Co-culture of Cbl-b−/− macrophages with 3T3-L1 adipocytes induced leptin expression and dephosphorylation of insulin receptor substrate 1, leading to impaired glucose uptake in adipocytes. Furthermore, Vav1, a key factor in macrophage activation, was highly phosphorylated in peritoneal Cbl-b−/− macrophages compared with Cbl-b+/+ macrophages. Treatment with a neutralizing anti–MCP-1 antibody improved peripheral insulin resistance and macrophage infiltration into WAT in elderly Cbl-b−/− mice.

CONCLUSIONS—Cbl-b is a negative regulator of macrophage infiltration and activation, and macrophage activation by Cbl-b deficiency contributes to the peripheral insulin resistance and glucose intolerance via cytokines secreted from macrophages.
The Peroxisome Proliferator–Activated Receptor-γ Agonist Pioglitazone Increases Number and Function of Endothelial Progenitor Cells in Patients With Coronary Artery Disease and Normal Glucose Tolerance
Authors: Werner Christian , Kamani Christel Hermann , Gensch Christoph , Böhm Michael , Laufs Ulrich
Published: September 2007

OBJECTIVE—Peroxisome proliferator–activated receptor-γ (PPARγ) agonists (thiazolidinediones [TZDs]) are used for the treatment of diabetes. Bone marrow–derived endothelial progenitor cells (EPCs) improve vascular function and predict cardiovascular risk. The effect of pioglitazone therapy on EPCs was examined.

RESEARCH DESIGN AND METHODS AND RESULTS—We performed a prospective, randomized, double-blind study on patients with documented stable coronary artery disease and normal glucose tolerance. Of 54 patients with normal fasting glucose levels, 18 showed impaired glucose tolerance and 36 patients with normal glucose tolerance were randomized to 30-day treatment with pioglitazone (45 mg) or placebo in addition to optimal medical therapy. All patients in the TZD group showed an increase of adiponectin levels as an indicator of compliance (11.4 ± 1.1 to 36.8 ± 2.1 μg/ml; P < 0.001). TZD, but not placebo, decreased mean high-sensitivity C-reactive protein to 43 ± 19% (P < 0.05). Pioglitazone increased CD34+/kinase insert domain receptor+ EPCs to 142 ± 9% and cultured 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine–labeled acetylated LDL+/lectin+ EPCs to 180 ± 3% (P < 0.05). EPC numbers were not changed in the placebo group. TZD increased the SDF-1–induced migratory capacity to 146 ± 9% per EPC number (P < 0.05) and upregulated the clonogenic potential of EPCs, increasing the colony-forming units to 172 ± 12% (P < 0.001). In cultured human EPCs, TZD increased EPC numbers and migration and reduced NADPH-oxidase activity. The TZD effect was reversed by the PPARγ antagonist GW9662 and mimicked by treatment with adiponectin.

CONCLUSIONS—The PPARγ agonist pioglitazone increases the number and function of EPCs in patients with coronary artery disease. The effect represents a potential regenerative mechanism in atherosclerosis and is observed in normoglycemic individuals with stable coronary artery disease.
The Role of Melanocortin 3 Receptor Gene in Childhood Obesity
Authors: Seng Lee Yung , Kok Seng Poh Larry , Lay Kee Kek Betty , Yin Loke Kah
Published: September 2007

OBJECTIVE—Melanocortin 3 receptor (MC3R) plays a critical role in weight regulation of rodents, but its role in humans remains unclear. The objective of this study was to identify genetic variants of the MC3R gene and determine its association with childhood obesity.

RESEARCH DESIGN AND METHODS—We screened 201 obese children for MC3R gene mutations with anthropometric measurements, blood tests, feeding behavior, and body composition assessment. We identified three novel heterozygous mutations (Ile183Asn, Ala70Thr, and Met134Ile) in three unrelated subjects, which were not found in 188 control subjects, and two common polymorphisms Thr6Lys and Val81Ile.

RESULTS—In vitro functional studies of the resultant mutant receptors revealed impaired signaling activity but normal ligand binding and cell surface expression. The heterozygotes demonstrated higher leptin levels and adiposity and less hunger compared with obese control subjects, reminiscent of the MC3R knockout mice. Family studies showed that these mutations may be associated with childhood or early-onset obesity. The common variants Thr6Lys and Val81Ile were in complete linkage disequilibrium, and in vitro studies revealed reduced signaling activity compared with wild-type MC3R. Obese subjects with the 6Lys/81Ile haplotype had significantly higher leptin levels, percentage body fat, and insulin sensitivity, and the causative role of the 6Lys/81Ile variants is supported by the presence of an additive effect in which heterozygotes had an intermediate phenotype compared with homozygotes.

CONCLUSIONS— MC3R mutations may not result in autosomal dominant forms of obesity but may contribute as a predisposing factor to childhood obesity and exert an effect on the human phenotype. Our report supports the role of MC3R in human weight regulation.
Evidence for a Role of Superoxide Generation in Glucose-Induced β-Cell Dysfunction In Vivo
Authors: Tang Christine , Han Ping , Oprescu Andrei I. , Lee Simon C. , Gyulkhandanyan Armen V. , Chan Gary N.Y. , Wheeler Michael B. , Giacca Adria
Published: September 2007

OBJECTIVE— Prolonged elevation of glucose can adversely affect β-cell function. In vitro studies have linked glucose-induced β-cell dysfunction to oxidative stress; however, whether oxidative stress plays a role in vivo is unclear. Therefore, our objective was to investigate the role of oxidative stress in an in vivo model of glucose-induced β-cell dysfunction.

RESEARCH DESIGN AND METHODS— Wistar rats were infused intravenously with glucose for 48 h to achieve 20 mmol/l hyperglycemia with/without co-infusion of one of the following antioxidants: taurine (2-amino ethanesulfonic acid) (TAU), an aldehyde scavenger; N-acetylcysteine (NAC), a precursor of glutathione; or tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) (TPO), a superoxide dismutase mimetic. This was followed by islet isolation or hyperglycemic clamp.

RESULTS— A 48-h glucose infusion decreased glucose-stimulated insulin secretion (GSIS) and elevated reactive oxygen species (ROS), total superoxide, and mitochondrial superoxide in freshly isolated islets. TPO prevented the increase in total and mitochondrial superoxide and the β-cell dysfunction induced by high glucose. However, TAU and NAC, despite completely normalizing H2DCF-DA (dihydro-dichlorofluorescein diacetate)-measured ROS, did not prevent the increase in superoxide and the decrease in β-cell function induced by high glucose. TPO but not TAU also prevented β-cell dysfunction induced by less extreme hyperglycemia (15 mmol/l) for a longer period of time (96 h). To further investigate whether TPO is effective in vivo, a hyperglycemic clamp was performed. Similar to the findings in isolated islets, prolonged glucose elevation (20 mmol/l for 48 h) decreased β-cell function as assessed by the disposition index (insulin secretion adjusted for insulin sensitivity), and co-infusion of TPO with glucose completely restored β-cell function.

CONCLUSIONS— These findings implicate superoxide generation in β-cell dysfunction induced by prolonged hyperglycemia.
In Vivo Cytotoxicity of Insulin-Specific CD8+ T-Cells in HLA-A*0201 Transgenic NOD Mice
Authors: Jarchum Irene , Baker Jason C. , Yamada Tatsuya , Takaki Toshiyuki , Marron Michele P. , Serreze David V. , DiLorenzo Teresa P.
Published: September 2007

OBJECTIVE—CD8+ T-cells specific for islet antigens are essential for the development of type 1 diabetes in the NOD mouse model of the disease. Such T-cells can also be detected in the blood of type 1 diabetic patients, suggesting their importance in the pathogenesis of the human disease as well. The development of peptide-based therapeutic reagents that target islet-reactive CD8+ T-cells will require the identification of disease-relevant epitopes.

RESEARCH DESIGN AND METHODS—We used islet-infiltrating CD8+ T-cells from HLA-A*0201 transgenic NOD mice in an interferon-γ enzyme-linked immunospot assay to identify autoantigenic peptides targeted during the spontaneous development of disease. We concentrated on insulin (Ins), which is a key target of the autoimmune response in NOD mice and patients alike.

RESULTS—We found that HLA-A*0201-restricted T-cells isolated from the islets of the transgenic mice were specific for Ins1 L3–11, Ins1 B5–14, and Ins1/2 A2–10. Insulin-reactive T-cells were present in the islets of mice as young as 5 weeks of age, suggesting an important function for these specificities early in the pathogenic process. Although there was individual variation in peptide reactivity, Ins1 B5–14 and Ins1/2 A2–10 were the immunodominant epitopes. Notably, in vivo cytotoxicity to cells bearing these peptides was observed, further confirming them as important targets of the pathogenic process.

CONCLUSIONS—The human versions of B5–14 and A2–10, differing from the murine peptides by only a single residue, represent excellent candidates to explore as CD8+ T-cell targets in HLA-A*0201–positive type 1 diabetic patients.
Mice Heterozygous for Tumor Necrosis Factor-α Converting Enzyme Are Protected From Obesity-Induced Insulin Resistance and Diabetes
Authors: Serino Matteo , Menghini Rossella , Fiorentino Loredana , Amoruso Roberta , Mauriello Alessandro , Lauro Davide , Sbraccia Paolo , Hribal Marta L. , Lauro Renato , Federici Massimo
Published: September 2007

OBJECTIVE—Tumor necrosis factor (TNF)-α is known to affect insulin sensitivity, glucose, and lipid metabolism through alternative and redundant mechanisms at both translational and post-translational levels. TNF-α exerts its paracrine effects once the membrane-anchored form is shed and released from the cell membrane. TNF-α cleavage is regulated by TNF-α converting enzyme (TACE), which regulates the function of several transmembrane proteins, such as interleukin-6 receptor and epidermal growth factor receptor ligands. The role of TACE in high-fat diet (HFD)-induced obesity and its metabolic complications is unknown.

RESEARCH DESIGN AND METHODS—To gain insights into the role of TACE in metabolic disorders, we used Tace+/− mice fed a standard or high-fat diet for 16 weeks.

RESULTS—We observed that Tace+/− mice are relatively protected from obesity and insulin resistance compared with wild-type littermates. When fed an HFD, wild-type mice exhibited visceral obesity, increased free fatty acid and monocyte chemoattractant protein (MCP)1 levels, hypoadiponectinemia, glucose intolerance, and insulin resistance compared with Tace+/− mice. Interestingly, Tace+/− mice exhibited increased uncoupling protein-1 and GLUT4 expression in white adipose tissue.

CONCLUSIONS—Our results suggest that modulation of TACE activity is a new pathway to be investigated for development of agents acting against obesity and its metabolic complications.