bluuebunny/crossref_metadata_2025 huggingface dataset:
Erratum to: Assessment of angle velocity in girls with adolescent idiopathic scoliosis
Authors: Escalada Ferran , Marco Ester , Belmonte Roser , Duarte Esther , Ma Muniesa Josep , Boza Roser , Tejero Marta , Cáceres Enric
Published: October 2009

Correction to Escalada F, Marco E, Duarte E, Muniesa JM, Boza R, Tejero M, Cáceres E. Assessment of angle velocity in girls with adolescent idiopathic scoliosis. Scoliosis 2009; 4:20.
In Vivo Characterization of High Basal Signaling from the Ghrelin Receptor
Authors: Petersen Pia Steen , Woldbye David P. D. , Madsen Andreas Nygaard , Egerod Kristoffer L. , Jin Chunyu , Lang Manja , Rasmussen Maria , Beck-Sickinger Annette G. , Holst Birgitte
Published: October 2009

The receptor for the orexigenic peptide, ghrelin, is one of the most constitutively active 7TM receptors known, as demonstrated under in vitro conditions. Change in expression of a constitutively active receptor is associated with change in signaling independent of the endogenous ligand. In the following study, we found that the expression of the ghrelin receptor in the hypothalamus was up-regulated approximately 2-fold in rats both during 48-h fasting and by streptozotocin-induced hyperphagia. In a separate experiment, to probe for the effect of the high basal signaling of the ghrelin receptor in vivo, we used intracerebroventricular administration by osmotic pumps of a peptide [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P. This peptide selectively displays inverse agonism at the ghrelin receptor as compared with an inactive control peptide with just a single amino acid substitution. Food intake and body weight were significantly decreased in the group of rats treated with the inverse agonist, as compared with the groups treated with the control peptide or the vehicle. In the hypothalamus, the expression of neuropeptide Y and uncoupling protein 2 was decreased by the inverse agonist. In a hypothalamic cell line that endogenously expresses the ghrelin receptor, we observed high basal activity of the cAMP response element binding protein, an important signaling transduction pathway for appetite regulation. The activation was further increased by ghrelin administration and decreased by administration of the inverse agonist. It is suggested that the high constitutive signaling activity is important for the in vivo function of the ghrelin receptor in the control of food intake and body weight.
Residues K128, 132, and 134 in the Thyroid Hormone Receptor-α Are Essential for Receptor Acetylation and Activity
Authors: Sánchez-Pacheco Aurora , Martínez-Iglesias Olaia , Méndez-Pertuz Marinela , Aranda Ana
Published: October 2009

The thyroid hormone receptor (TR)-α is a nuclear receptor that mediates both transrepression and ligand-dependent transactivation. Here we show that TRα is posttranslationally modified by acetylation in response to its own ligand (T3). Acetylation increases binding to DNA. Using mutagenesis, we identified three conserved lysine residues in the carboxi-terminal extension (CTE) of the DNA binding domain that are targets of the cAMP-response element-binding protein acetyltransferase. Substitution of these lysines by arginines in TRα decreased ligand binding affinity and precluded ligand-dependent release of corepressors and recruitment of coactivators. The acetylation TRα mutant lost the ability to transactivate even at high T3 concentrations and acts as a dominant-negative inhibitor of wild-type TR activity. In addition, whereas native TRα interferes with AP-1 function, the mutant is unable to mediate transrepression. Finally, TRα suppresses NIH-3T3 fibroblast transformation by the Ras oncogene both in a ligand-dependent and -independent manner, but the CTE mutant is unable to mediate ligand-dependent repression of transformation. These results reveal a key role for the CTE region on acetylation, ligand affinity, transactivation, transrepression, and antitransforming properties of TRα.
Impaired Skeletal Muscle β-Adrenergic Activation and Lipolysis Are Associated with Whole-Body Insulin Resistance in Rats Bred for Low Intrinsic Exercise Capacity
Authors: Lessard Sarah J. , Rivas Donato A. , Chen Zhi-Ping , van Denderen Bryce J. , Watt Matthew J. , Koch Lauren G. , Britton Steven L. , Kemp Bruce E. , Hawley John A.
Published: October 2009

Rats selectively bred for high endurance running capacity (HCR) have higher insulin sensitivity and improved metabolic health compared with those bred for low endurance capacity (LCR). We investigated several skeletal muscle characteristics, in vitro and in vivo, that could contribute to the metabolic phenotypes observed in sedentary LCR and HCR rats. After 16 generations of selective breeding, HCR had approximately 400% higher running capacity (P < 0.001), improved insulin sensitivity (P < 0.001), and lower fasting plasma glucose and triglycerides (P < 0.05) compared with LCR. Skeletal muscle ceramide and diacylglycerol content, basal AMP-activated protein kinase (AMPK) activity, and basal lipolysis were similar between LCR and HCR. However, the stimulation of lipolysis in response to 10 μm isoproterenol was 70% higher in HCR (P = 0.004). Impaired isoproterenol sensitivity in LCR was associated with lower basal triacylglycerol lipase activity, Ser660 phosphorylation of HSL, and β2-adrenergic receptor protein content in skeletal muscle. Expression of the orphan nuclear receptor Nur77, which is induced by β-adrenergic signaling and is associated with insulin sensitivity, was lower in LCR (P < 0.05). Muscle protein content of Nur77 target genes, including uncoupling protein 3, fatty acid translocase/CD36, and the AMPK γ3 subunit were also lower in LCR (P < 0.05). Our investigation associates whole-body insulin resistance with impaired β-adrenergic response and reduced expression of genes that are critical regulators of glucose and lipid metabolism in skeletal muscle. We identify impaired β-adrenergic signal transduction as a potential mechanism for impaired metabolic health after artificial selection for low intrinsic exercise capacity.
Characterization of Conserved Tandem Donor Sites and Intronic Motifs Required for Alternative Splicing in Corticosteroid Receptor Genes
Authors: Rivers Caroline , Flynn Andrea , Qian Xiaoxiao , Matthews Laura , Lightman Stafford , Ray David , Norman Michael
Published: October 2009

Alternative splicing events from tandem donor sites result in mRNA variants coding for additional amino acids in the DNA binding domain of both the glucocorticoid (GR) and mineralocorticoid (MR) receptors. We now show that expression of both splice variants is extensively conserved in mammalian species, providing strong evidence for their functional significance. An exception to the conservation of the MR tandem splice site (an A at position +5 of the MR+12 donor site in the mouse) was predicted to decrease U1 small nuclear RNA binding. In accord with this prediction, we were unable to detect the MR+12 variant in this species. The one exception to the conservation of the GR tandem splice site, an A at position +3 of the platypus GRγ donor site that was predicted to enhance binding of U1 snRNA, was unexpectedly associated with decreased expression of the variant from the endogenous gene as well as a minigene. An intronic pyrimidine motif present in both GR and MR genes was found to be critical for usage of the downstream donor site, and overexpression of TIA1/TIAL1 RNA binding proteins, which are known to bind such motifs, led to a marked increase in the proportion of GRγ and MR+12. These results provide striking evidence for conservation of a complex splicing mechanism that involves processes other than stochastic spliceosome binding and identify a mechanism that would allow regulation of variant expression.
Short-Term Regulation of Tumor Necrosis Factor-α-Induced Lipolysis in 3T3-L1 Adipocytes Is Mediated through the Inducible Nitric Oxide Synthase/Nitric Oxide-Dependent Pathway
Authors: Lien Chih-Chan , Au Lo-Chun , Tsai Ying-Lan , Ho Low-Tone , Juan Chi-Chang
Published: October 2009

TNF-α has several effects on adipocytes that may be related to the development of type 2 diabetes in obese subjects. Many studies demonstrated that long-term treatment with TNF-α increases lipolysis in adipocytes. However, the short-term (<4 h) effects of TNF-α on lipolysis have not been well investigated. The aim of this study was to investigate the short-term regulatory mechanism of TNF-α-induced lipolysis in 3T3-L1 adipocytes. Well-differentiated 3T3-L1 adipocytes were used. Lipolysis was determined by measuring glycerol release. Expression of inducible nitric oxide (iNOS) and nitric oxide (NO) production were measured, respectively, by Western blots and the Griess reagent. A selective iNOS inhibitor (s-ethylisothiourea · HBr), an adenylyl cyclase inhibitor (SQ22536), and a guanylyl cyclase inhibitor (LY83583) were used to investigate the involvement of iNOS, cAMP, and cGMP in TNF-α-induced lipolysis. Transient transfection with iNOS short hairpin RNA was performed to confirm the involvement of iNOS in TNF-α-induced lipolysis. Phosphorylation of hormone-sensitive lipase (HSL) was measured by immunoprecipitation and Western blotting. Results showed that short-term TNF-α treatment significantly increased lipolysis, iNOS expression, and NO production in a time- and dose-dependent manner. Furthermore, treatment with the NO donor S-nitroso-N-acetylpenicillamine also stimulated lipolysis and HSL phosphorylation in 3T3-L1 adipocytes. Moreover, pretreatment with inhibitors of iNOS and guanylate cyclase, but not an adenylate cyclase inhibitor, abolished TNF-α-induced lipolysis and HSL phosphorylation. Suppression of TNF-α-induced iNOS expression using short hairpin RNA significantly reduced TNF-α-induced lipolysis. In conclusion, short-term TNF-α treatment induces lipolysis in 3T3-L1 adipocytes by increasing iNOS expression and NO production, which activates the guanylyl cyclase/cGMP-dependent pathway and induces phosphorylation of HSL.
Melatonin Improves Glucose Homeostasis and Endothelial Vascular Function in High-Fat Diet-Fed Insulin-Resistant Mice
Authors: Sartori Claudio , Dessen Pierre , Mathieu Caroline , Monney Anita , Bloch Jonathan , Nicod Pascal , Scherrer Urs , Duplain Hervé
Published: October 2009

Obesity and insulin resistance represent a problem of utmost clinical significance worldwide. Insulin-resistant states are characterized by the inability of insulin to induce proper signal transduction leading to defective glucose uptake in skeletal muscle tissue and impaired insulin-induced vasodilation. In various pathophysiological models, melatonin interacts with crucial molecules of the insulin signaling pathway, but its effects on glucose homeostasis are not known. In a diet-induced mouse model of insulin resistance and normal chow-fed control mice, we sought to assess the effects of an 8-wk oral treatment with melatonin on insulin and glucose tolerance and to understand underlying mechanisms. In high-fat diet-fed mice, but not in normal chow-fed control mice, melatonin significantly improved insulin sensitivity and glucose tolerance, as evidenced by a higher rate of glucose infusion to maintain euglycemia during hyperinsulinemic clamp studies and an attenuated hyperglycemic response to an ip glucose challenge. Regarding underlying mechanisms, we found that melatonin restored insulin-induced vasodilation to skeletal muscle, a major site of glucose utilization. This was due, at least in part, to the improvement of insulin signal transduction in the vasculature, as evidenced by increased insulin-induced phosphorylation of Akt and endoethelial nitric oxide synthase in aortas harvested from melatonin-treated high-fat diet-fed mice. In contrast, melatonin had no effect on the ability of insulin to promote glucose uptake in skeletal muscle tissue in vitro. These data demonstrate for the first time that in a diet-induced rodent model of insulin resistance, melatonin improves glucose homeostasis by restoring the vascular action of insulin.
Global Disturbances in Autonomic Function Yield Cardiovascular Instability and Hypertension in the Chromogranin A Null Mouse
Authors: Gayen Jiaur R. , Gu Yusu , O'Connor Daniel T. , Mahata Sushil K.
Published: October 2009

We reported previously that chromogranin A (Chga) knockout (KO) mice are hypertensive and hyperadrenergic. Here we sought to determine the basis of such alterations by probing physiological, biochemical, and pharmacological responses to perturbations of the autonomic nervous system. In the conscious state, KO mice had substantially elevated basal high blood pressure (BP) and heart rate (HR); immobilization stress caused increments in systolic BP and HR in both wild-type (WT) and KO mice, with higher maxima but blunted increments in the KO state. Catestatin (CST; CHGA352–372) selectively diminished stress-induced increments in BP and HR in KO mice, implicating CST as an antihypertensive peptide, even in stressful conditions. Heightened plasma catecholamines in KO mice returned to WT level after CST. Stress caused further increments in catecholamines in WT mice but no change in KO mice. KO mice displayed diminished baroreflex sensitivity in response to either phenylephrine or sodium nitroprusside, accounting for exaggerated pressor and depressor responses to these compounds; baroreceptor function was normalized by CST. To probe the relative roles of endogenous/basal sympathetic vs. parasympathetic tone in control of BP and HR, we used the muscarinic-cholinergic antagonist atropine or the β-adrenergic antagonist propranolol; HR and BP responses to each antagonist were exaggerated in KO animals. We conclude that ablation of Chga expression results in global disturbances in autonomic function, both sympathetic and parasympathetic, that can be abrogated (or rescued), at least in part, by replacement of CST. The results point to mechanisms whereby CHGA and its CST fragment act to control cardiovascular homeostasis.
Accelerated Cartilage Resorption by Chondroclasts during Bone Fracture Healing in Osteoprotegerin-Deficient Mice
Authors: Ota Norikazu , Takaishi Hironari , Kosaki Naoto , Takito Jiro , Yoda Masaki , Tohmonda Takahide , Kimura Tokuhiro , Okada Yasunori , Yasuda Hisataka , Kawaguchi Hiroshi , Matsumoto Morio , Chiba Kazuhiro , Ikegami Hiroyasu , Toyama Yoshiaki
Published: October 2009

Receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG), a decoy receptor of RANKL, maintain bone mass by regulating the differentiation of osteoclasts, which are bone-resorbing cells. Endochondral bone ossification and bone fracture healing involve cartilage resorption, a less well-understood process that is needed for replacement of cartilage by bone. Here we describe the role of OPG produced by chondrocytes in chondroclastogenesis. Fracture healing in OPG−/− mice showed faster union of the fractured bone, faster resorption of the cartilaginous callus, and an increased number of chondroclasts at the chondroosseous junctions compared with that in wild-type littermates. When a cultured pellet of OPG−/− chondrocytes was transplanted beneath the kidney capsule, the pellet recruited many chondroclasts. The pellet showed the ability to induce tartrate-resistant acid phosphatase-positive multinucleated cells from RAW 264.7 cells in vitro. Finally, OPG−/− chondrocytes (but not wild-type chondrocytes) cultured with spleen cells induced many tartrate-resistant acid phosphatase-positive multinucleated cells. The expression of RANKL and OPG in chondrocytes was regulated by several osteotropic factors including 1,25-dihydroxyvitamin D3, PTHrP, IL-1α, and TNF-α. Thus, local OPG produced by chondrocytes probably controls cartilage resorption as a negative regulator for chondrocyte-dependent chondroclastogenesis.
Phosphatidyl Inositol 3-Kinase Signaling in Hypothalamic Proopiomelanocortin Neurons Contributes to the Regulation of Glucose Homeostasis
Authors: Hill Jennifer W. , Xu Yong , Preitner Frederic , Fukuda Makota , Cho You-Ree , Luo Ji , Balthasar Nina , Coppari Roberto , Cantley Lewis C. , Kahn Barbara B. , Zhao Jean J. , Elmquist Joel K.
Published: October 2009

Recent studies demonstrated a role for hypothalamic insulin and leptin action in the regulation of glucose homeostasis. This regulation involves proopiomelanocortin (POMC) neurons because suppression of phosphatidyl inositol 3-kinase (PI3K) signaling in these neurons blunts the acute effects of insulin and leptin on POMC neuronal activity. In the current study, we investigated whether disruption of PI3K signaling in POMC neurons alters normal glucose homeostasis using mouse models designed to both increase and decrease PI3K-mediated signaling in these neurons. We found that deleting p85α alone induced resistance to diet-induced obesity. In contrast, deletion of the p110α catalytic subunit of PI3K led to increased weight gain and adipose tissue along with reduced energy expenditure. Independent of these effects, increased PI3K activity in POMC neurons improved insulin sensitivity, whereas decreased PI3K signaling resulted in impaired glucose regulation. These studies show that activity of the PI3K pathway in POMC neurons is involved in not only normal energy regulation but also glucose homeostasis.
Bone Formation Regulates Circulating Concentrations of Fibroblast Growth Factor 23
Authors: Samadfam Rana , Richard Christian , Nguyen-Yamamoto Loan , Bolivar Isabel , Goltzman David
Published: October 2009

We examined the role of bone remodeling in the regulation of circulating concentrations of FGF23 using mouse models manifesting differing degrees of coupled and uncoupled bone turnover. Administration of the antiresorptive agent osteoprotegerin produced a profound reduction in bone resorption and formation in male and oophorectomized female mice, accompanied by an increase in serum levels of fibroblast growth factor 23 (FGF23) and a reduction in circulating 1,25-dihydroxyvitamin D [1,25(OH)2D]. In contrast, exogenous PTH(1-34) administration increased bone turnover and reduced circulating FGF23. In 1,25(OH)2D-deficient, 25-hydroxyvitamin D 1α-hydroxylase null mice on a high-calcium diet, endogenous PTH was elevated, bone formation but not resorption was increased, and serum FGF23 was virtually undetectable; on a rescue diet, serum calcium was normalized, PTH levels were reduced, bone formation was reduced, and serum FGF23 levels increased. After PTH treatment of wild-type mice, gene expression of dentin matrix protein 1 (DMP1) in bone was increased, whereas gene expression of FGF23 was reduced. In vitro studies in the osteoblastic cell line UMR-106 showed that externally added DMP1 could inhibit FGF23 gene expression and production stimulated by 1,25(OH)2D3. The results show that osteoblastic bone formation is a potent modulator of FGF23 production and release into the circulation, suggest that the biological consequences on mineral homeostasis of circulating FGF23 may also be dependent on the prevailing rate of bone turnover, and provide evidence that DMP1 may be a direct negative regulator of FGF23 production in osteoblastic cells.
Maternal High-Fat Diet Promotes Body Length Increases and Insulin Insensitivity in Second-Generation Mice
Authors: Dunn Gregory A. , Bale Tracy L.
Published: October 2009

Maternal obesity and diet consumption during pregnancy have been linked to offspring adiposity, cardiovascular disease, and impaired glucose metabolism. Furthermore, nutrition during development is clearly linked to somatic growth. However, few studies have examined whether phenotypes derived from maternal high-fat diet exposure can be passed to subsequent generations and by what mechanisms this may occur. Here we report the novel finding of a significant body length increase that persisted across at least two generations of offspring in response to maternal high-fat diet exposure. This phenotype is not attributable to altered intrauterine conditions or maternal feeding behavior because maternal and paternal lineages were able to transmit the effect, supporting a true epigenetic manner of inheritance. We also detected a heritable feature of reduced insulin sensitivity across two generations. Alterations in the GH secretagogue receptor (GHSR), the GHSR transcriptional repressor AF5q31, plasma IGF-I concentrations, and IGF-binding protein-3 (IGFBP3) suggest a contribution of the GH axis. These studies provide evidence that the heritability of body length and glucose homeostasis are modulated by maternal diet across multiple generations, providing a mechanism where length can increase rapidly in concert with caloric availability.
Melanocortins May Stimulate Reproduction by Activating Orexin Neurons in the Dorsomedial Hypothalamus and Kisspeptin Neurons in the Preoptic Area of the Ewe
Authors: Backholer Kathryn , Smith Jeremy , Clarke Iain J.
Published: October 2009

To further test the hypothesis that melanocortins stimulate the reproductive axis, we treated ewes with melanocortin agonist (MTII) in the luteal phase of the estrous cycle and during seasonal anestrus. Lateral ventricular infusion of MTII (10 μg/h) during the luteal phase increased LH secretion. Retrograde neuronal tracing in the brain showed few proopiomelanocortin or kisspeptin cells in the arcuate nucleus, but more than 70% of kisspeptin cells in the dorsolateral preoptic area (POA), projecting to the ventromedial POA in which GnRH cells are located. MTII infusion (20 h) was repeated in luteal phase ewes and brains were harvested to measure gene expression of preproorexin and kisspeptin. Expression of orexin in the dorsomedial hypothalamus and kisspeptin in the POA was up-regulated by MTII treatment and Kiss1 in the arcuate nucleus was down-regulated. Seasonally anestrous ewes were progesterone primed and then treated (lateral ventricular) with MTII (10 μg/h) or vehicle for 30 h, and blood samples were collected every 2 h from 4 h before infusion until 6 h afterward to monitor acute response in terms of LH levels. A rise in basal LH levels was seen, but samples collected around the time of the predicted LH surge did not indicate that an ovulatory event occurred. We conclude that melanocortins are positive regulators of the reproductive neuroendocrine system, but treatment with melanocortins does not fully overcome seasonal acyclicity. The stimulatory effect of melanocortin in the luteal phase of the estrous cycle may be via the activation of kisspeptin cells in the POA and/or orexin cells in the dorsomedial hypothalamus.
Gonadotropin-Releasing Hormone Neuroterminals and Their Microenvironment in the Median Eminence: Effects of Aging and Estradiol Treatment
Authors: Yin Weiling , Wu Di , Noel Megan L. , Gore Andrea C.
Published: October 2009

The GnRH decapeptide controls reproductive function through its release from neuroendocrine terminals in the median eminence, a site where there is a convergence of numerous nerve terminals and glial cells. Previous work showed dynamic changes in the GnRH-glial-capillary network in the median eminence under different physiological conditions. Because aging in rats is associated with a diminution of GnRH release and responsiveness to estradiol feedback, we examined effects of age and estradiol treatment on these anatomical interactions. Rats were ovariectomized at young (4 months), middle-aged (11 months), or old (22–23 months) ages, allowed 4 wk to recover, and then treated with vehicle or estradiol for 72 h followed by perfusion. Immunofluorescence of GnRH was measured, and immunogold electron microscopic analyses were performed to study the ultrastructural properties of GnRH neuroterminals and their microenvironment. Although the GnRH immunofluorescent signal showed no significant changes with age and estradiol treatment, we found that the median eminence underwent both qualitative and quantitative structural changes with age, including a disorganization of cytoarchitecture with aging and a decrease in the apposition of GnRH neuroterminals to glia with age and estradiol treatment. Thus, although GnRH neurons can continue to synthesize and transport peptide, changes in the GnRH neuroterminal-glial-capillary machinery occur during reproductive senescence in a manner consistent with a disconnection of these elements and a potential dysregulation of GnRH neurosecretion.
Increased Tau Phosphorylation and Cleavage in Mouse Models of Type 1 and Type 2 Diabetes
Authors: Kim Bhumsoo , Backus Carey , Oh SangSu , Hayes John M. , Feldman Eva L.
Published: October 2009

As the population of the United States ages, the incidence of age-related neurodegenerative and systemic diseases including Alzheimer’s disease (AD) and diabetes is increasing rapidly. Multiple studies report that patients with diabetes have a 50–75% increased risk of developing AD compared with age- and gender-matched patients without diabetes. Abnormally phosphorylated tau is a major building block of neurofibrillary tangles, a classic neuropathological characteristic of AD. In addition, proteolytic tau cleavage promotes AD progression due to cleaved tau serving as a nucleation center for the pathological assembly of tau filaments. The current study examines tau modification in type 1 (streptozotocin-injected) and type 2 (db/db) mouse models of diabetes. Tau phosphorylation is increased in the cortex and hippocampus of db/db mice compared with db+ control mouse brain. Interestingly, there is an age-dependent increase in tau cleavage that is not observed in age-matched control db+ animals. Streptozotocin injection also increased tau phosphorylation; however, the increase was less significant compared with the type 2 mouse model, and more importantly, no tau cleavage was detected. Our results suggest tau modification caused by insulin dysfunction and hyperglycemia may contribute to the increased incidence of AD in diabetes. We hypothesize that type 1 and type 2 diabetes may contribute to AD through different mechanisms; in type 2 diabetes, hyperglycemia-mediated tau cleavage may be the key feature, whereas insulin deficiency may be the major contributing factor in type 1 diabetes.
Knockdown of the Type 3 Iodothyronine Deiodinase (D3) Interacting Protein Peroxiredoxin 3 Decreases D3-Mediated Deiodination in Intact Cells
Authors: Aerts Goele , Arrojo e Drigo Rafael , Van Herck Stijn L. J. , Sammels Eva , Mirebeau-Prunier Delphine , Gereben Balázs , Zeöld Anikó , Harney John W. , Huang Stephen A. , Mulcahey Michelle A. , Van der Geyten Serge , Van den Bergh Gert , Arckens Lut , Darras Veerle M. , Zavacki Ann Marie
Published: October 2009

The type 3 iodothyronine deiodinase (D3) is the primary deiodinase that inactivates thyroid hormone. Immunoprecipitation of D3, followed by fluorescent two-dimensional difference gel electrophoresis and mass spectrometry, identified peroxiredoxin 3 (Prx3) as a D3-associated protein. This interaction was confirmed using reverse coimmunoprecipitation, in which pull-down of Prx3 resulted in D3 isolation, and by fluorescence resonance energy transfer between cyan fluorescent protein-D3 and yellow fluorescent protein-Prx3. Prx3 overexpression did not change D3 activity in transfected HEK 293 cells; however, Prx3 knockdown resulted in a 50% decrease in D3-mediated whole-cell deiodination. Notably, D3 activity of cell lysates with dithiothreitol as an exogenous reducing factor and D3 protein levels were not decreased with Prx3 knockdown, indicating that the observed reduction in whole-cell deiodination was not simply due to a decrease in D3 enzyme levels. Prx3 knockdown did not change D3’s affinity for T3 because saturation of D3-mediated whole-cell deiodination occurred between 20 and 200 nm T3 both with and without Prx3. Furthermore, the decrease in D3 activity in whole cells was not attributable to nonspecific oxidative stress because pretreatment with the antioxidant N-acetyl cysteine did not reverse the effects of Prx3 knockdown. Thioredoxin, the cofactor needed for Prx3 regeneration, supported D3 microsomal activity; however, Prx3 knockdown did not change D3 activity in this system. In conclusion, knockdown of Prx3 decreases D3 activity in whole cells, whereas absolute levels of D3 are unchanged, consistent with Prx3 playing a rate-limiting role in the regeneration of the D3 enzyme.
Spatiotemporal Coupling of Focal Extracellular Matrix Degradation and Reconstruction in the Menstrual Human Endometrium
Authors: Gaide Chevronnay Héloïse P. , Galant Christine , Lemoine Pascale , Courtoy Pierre J. , Marbaix Etienne , Henriet Patrick
Published: October 2009

Coupling of focal degradation and renewal of the functional layer of menstrual endometrium is a key event of the female reproductive biology. The precise mechanisms by which the various endometrial cell populations control extracellular matrix (ECM) degradation in the functionalis while preserving the basalis and the respective contribution of basalis and functionalis in endometrium regeneration are still unclear. We therefore compared the transcriptome of stromal and glandular cells isolated by laser capture microdissection from the basalis as well as degraded and preserved areas of the functionalis in menstrual endometria. Data were validated by in situ hybridization. Expression profile of selected genes was further analyzed throughout the menstrual cycle, and their response to ovarian steroids withdrawal was studied in a mouse xenograft model. Immunohistochemistry confirmed the results at the protein level. Algorithms for sample clustering segregated biological samples according to cell type and tissue depth, indicating distinct gene expression profiles. Pairwise comparisons identified the greatest numbers of differentially expressed genes in the lysed functionalis when compared with the basalis. Strikingly, in addition to genes products associated with tissue degradation (matrix metalloproteinase and plasmin systems) and apoptosis, superficial lysed stroma was enriched in gene products associated with ECM biosynthesis (collagens and their processing enzymes). These results support the hypothesis that fragments of the functionalis participate in endometrial regeneration during late menstruation. Moreover, menstrual reflux of lysed fragments overexpressing ECM components and adhesion molecules could easily facilitate implantation of endometriotic lesions.
The Blood-Brain Barrier Thyroxine Transporter Organic Anion-Transporting Polypeptide 1c1 Displays Atypical Transport Kinetics
Authors: Westholm Daniel E. , Salo David R. , Viken Kevin J. , Rumbley Jon N. , Anderson Grant W.
Published: October 2009

Organic anion-transporting polypeptide (Oatp) 1c1 is a high-affinity T4 transporter expressed in brain barrier cells. Oatp1c1 transports a variety of additional ligands including the conjugated sterol estradiol 17β-glucuronide (E217βG). Intriguingly, published data suggest that E217βG inhibition of Oatp1c1-mediated T4 transport exhibits characteristics suggestive of atypical transport kinetics. To determine whether Oatp1c1 exhibits atypical transport kinetics, we first performed detailed T4 and E217βG uptake assays using Oatp1c1 stably transfected HEK293 cells and a wide range of T4 and E217βG concentrations (100 pm to 300 nm and 27 nm to 200 μm, respectively). Eadie-Hofstee plots derived from these detailed T4 and E217βG uptake experiments display a biphasic profile consistent with atypical transport kinetics. These data along with T4 and E217βG cis-inhibition dose-response measurements revealed shared high- and low-affinity Oatp1c1 binding sites for T4 and E217βG. T4 and E217βG recognized these Oatp1c1 binding sites with opposite preferences. In addition, sterols glucuronidated in the 17 or 21 position, exhibited preferential substrate-dependent inhibition of Oatp1c1 transport, inhibiting Oatp1c1-mediated E217βG transport more strongly than T4 transport. Together these data reveal that Oatp1c1-dependent substrate transport is a complex process involving substrate interaction with multiple binding sites and competition for binding with a variety of other substrates. A thorough understanding of atypical Oatp1c1 transport processes and substrate-dependent inhibition will allow better prediction of endo- and xenobiotic interactions with the Oatp transporter.
Fingers as a Marker of Prenatal Androgen Exposure
Authors: Berenbaum Sheri A. , Bryk Kristina Korman , Nowak Nicole , Quigley Charmian A. , Moffat Scott
Published: October 2009

Interest in biological substrates of sex-related variations in psychological and physiological characteristics has led to a search for biomarkers of prenatal hormone exposure that can be measured postnatally. There has been particular interest in digit ratio, the relative lengths of the second and fourth fingers (2D:4D), but its validity as a measure of prenatal androgen has not been established. We report the strongest evaluation of the value of 2D:4D as a biomarker for early androgen exposure. Individuals with 46,XY karyotype but no effective prenatal androgen exposure due to complete androgen insensitivity syndrome had digit ratios that were feminized: they were higher than those of typical men and similar to those of typical women. Nevertheless, the effect was modest in size, and there was considerable within-group variability and between-group overlap, indicating that digit ratio is not a good marker of individual differences in prenatal androgen exposure.
Protection against Loss of Innate Defenses in Adulthood by Low Advanced Glycation End Products (AGE) Intake: Role of the Antiinflammatory AGE Receptor-1
Authors: Vlassara Helen , Cai Weijing , Goodman Susan , Pyzik Renata , Yong Angie , Chen Xue , Zhu Li , Neade Tina , Beeri Michal , Silverman Jeremy M. , Ferrucci Luigi , Tansman Laurie , Striker Gary E. , Uribarri Jaime
Published: October 2009

Context: Increased oxidant stress and inflammation (OS/infl) are linked to both aging-related diseases and advanced glycation end products (AGEs). Whereas AGE receptor-1 (AGER1) reduces OS/infl in animals, this has not been assessed in normal humans.

Objective: The objectives of the study were to determine whether AGER1 correlates with AGEs and OS/infl and a reduction of dietary AGEs (dAGEs) lowers OS/infl in healthy adults and chronic kidney disease (CKD-3) patients.

Design: This study was cross-sectional with 2-yr follow-up studies of healthy adults and CKD-3 patients, a subset of which received a reduced AGE or regular diet.

Setting: The study was conducted at general community and renal clinics.

Participants: Participants included 325 healthy adults (18–45 and >60 yr old) and 66 CKD-3 patients.

Intervention: An isocaloric low-AGE (30–50% reduction) or regular diet was given to 40 healthy subjects for 4 months and to nine CKD-3 patients for 4 wk.

Main Outcome: Relationships between age, dAGEs, serum AGEs, peripheral mononuclear cell AGE-receptors, and OS/Infl before and after reduction of dAGE intake were measured.

Results: AGEs, oxidant stress, receptor for AGE, and TNFα were reduced in normal and CKD-3 patients after the low-AGE diet, independently of age. AGER1 levels in CKD-3 patients on the low-AGE diet resembled 18- to 45-yr-old normal subjects. Dietary, serum, and urine AGEs correlated positively with peripheral mononuclear cell AGER1 levels in healthy participants. AGER1 was suppressed in CKD-3 subjects, whereas receptor for AGE and TNFα were increased.

Conclusions: Reduction of AGEs in normal diets may lower oxidant stress/inflammation and restore levels of AGER1, an antioxidant, in healthy and aging subjects and CKD-3 patients. AGE intake has implications for health outcomes and costs and warrants further testing.

Reduction of advanced glycation endproducts (AGE) in normal diets lowers oxidant stress/inflammation, and restores levels of AGE receptor-1 in healthy, aging, and chronic kidney disease-3 patients.
Impact of Growth Hormone Receptor Blockade on Substrate Metabolism during Fasting in Healthy Subjects
Authors: Moller Louise , Norrelund Helene , Jessen Niels , Flyvbjerg Allan , Pedersen Steen B. , Gaylinn Bruce D. , Liu Jianhua , Thorner Michael O. , Moller Niels , Lunde Jorgensen Jens Otto
Published: October 2009

Context: Experimental studies in GH-deficient patients and in healthy subjects receiving somatostatin-infusion suggest that GH is an important regulator of substrate metabolism during fasting. These models may not adequately reflect the selective effects of GH, and GH receptor (GHR) blockade offers a new model to define the metabolic role of GH.

Objective: The aim of this study was to investigate the impact of GHR blockade on substrate metabolism and insulin sensitivity during fasting.

Design: We conducted a randomized, placebo-controlled, crossover study in 10 healthy young men.

Intervention: After 36 h of fasting with saline or pegvisomant (GHR blockade), the subjects were studied during a 4-h basal period and 2.5-h hyperinsulinemic euglycemic clamp.

Main Outcome: We measured whole-body and forearm glucose, lipid, and protein metabolism, peripheral insulin sensitivity, and acyl and desacyl ghrelin.

Results: GHR blockade significantly suppressed circulating free fatty acids (1226 ± 83 vs. 1074 ± 65 μmol/liter; P = 0.03) and ketone bodies (3080 ± 271 vs. 2015 ± 235 μmol/liter; P ≤ 0.01), as well as forearm uptake of free fatty acids (0.341 ± 0.150 vs. 0.004 ± 0.119 μmol/100 ml · min; P < 0.01) and lipid oxidation (1.3 ± 0.1 vs. 1.2 ± 0.1 mg/kg · min; P = 0.03) in the basal period. By contrast, IGF-I levels in either serum or peripheral tissues were not impacted by GHR blockade, and protein metabolism was also unaffected. Basal glucose levels were elevated by GHR blockade, but insulin sensitivity was similar; this was associated with an increased acyl/desacyl ghrelin ratio.

Conclusion: GHR blockade, without changes in circulating or tissue IGF-I levels, selectively suppresses lipid mobilization and oxidation after short-term fasting. This supports the notion that stimulation of lipolysis is a primary and important effect of GH.

GH receptor blockade during fasting in healthy subjects suppresses lipid metabolism without a change in insulin sensitivity or protein metabolism.
Novel LMF1 Nonsense Mutation in a Patient with Severe Hypertriglyceridemia
Authors: Cefalù Angelo B. , Noto Davide , Arpi Maria Luisa , Yin Fen , Spina Rossella , Hilden Hannele , Barbagallo Carlo M. , Carroccio Antonio , Tarugi Patrizia , Squatrito Sebastiano , Vigneri Riccardo , Taskinen Marja-Riitta , Péterfy Miklós , Averna Maurizio R.
Published: October 2009

Context: Lipase maturation factor 1 (LMF1) gene is a novel candidate gene in severe hypertriglyceridemia. Lmf1 is involved in the maturation of lipoprotein lipase (LPL) and hepatic lipase in endoplasmic reticulum. To date only one patient with severe hypertriglyceridemia and related disorders was found to be homozygous for a nonsense mutation in LMF1 gene (Y439X).

Objective: The objective of the study was to investigate LMF1 gene in hypertriglyceridemic patients in whom mutations in LPL, APOC2, and APOA5 genes had been excluded.

Results: The resequencing of LMF1 gene led to the discovery of a novel homozygous nonsense mutation in one patient with severe hypertriglyceridemia and recurrent episodes of pancreatitis. The mutation causes a G>A substitution in exon 9 (c.1395G>A), leading to a premature stop codon (W464X). LPL activity and mass were reduced by 76 and 50%, respectively, compared with normolipidemic controls. The proband over the years has shown a good response to treatment. The proband’s son, heterozygous for the W464X, shows normal plasma triglyceride levels.

Conclusions: We identified the second novel pathogenic mutation in LMF1 gene in a patient with severe hypertriglyceridemia. LPL deficiency in our patient was milder than in the carrier of the Y439X previously described.

Identification and functional analysis of a novel nonsense mutation of the LMF1 gene in a patient with severe hypertriglyceridemia.
Association of Variation in the Interleukin-1 Gene Family with Diabetes and Glucose Homeostasis
Authors: Luotola Kari , Pääkkönen Rauni , Alanne Mervi , Lanki Timo , Moilanen Leena , Surakka Ida , Pietilä Arto , Kähönen Mika , Nieminen Markku S. , Kesäniemi Y. Antero , Peters Annette , Jula Antti , Perola Markus , Salomaa Veikko ,   for the Health 2000 AIRGENE Study Groups
Published: October 2009

Objective: Proinflammatory cytokine IL-1β is capable of decreasing insulin-induced glucose transport. Therefore, we hypothesized that genetic variation in the IL-1 gene family is associated with measures of glucose homeostasis and diabetes.

Design and Outcome Measures: Fifteen haplotype-tagging single-nucleotide polymorphisms in the IL-1α, IL-1β, and IL-1 receptor antagonist genes were determined in a Finnish population survey (n = 6771). Glucose and insulin concentrations were measured, and indices of insulin resistance and β-cell function were calculated using the homeostasis model assessment. Two-hour oral glucose tolerance tests were carried out on a subsample of 1390 participants. Associations with prevalent diabetes were tested for replication in a sample of European myocardial infarction survivors (n = 972).

Results: The minor allele of the IL-1β rs1143634(G→A) was associated with higher blood glucose than the major allele: 5.37, 5.41, and 5.48 mmol/liter for the GG, AG, and AA genotypes, respectively (multivariate adjusted P for trend <0.0001; Bonferroni corrected P = 0.00096). The 2-h glucose was also higher (6.45 and 7.20 mmol/liter for the GG vs. AA; P = 0.003, Bonferroni corrected P = 0.045). The haplotype ACG of rs1143634, rs3917356, and rs16944 associated with higher glucose, higher homeostasis model assessment for insulin resistance index, higher 2-h insulin, and prevalent diabetes (adjusted rate ratio = 1.54; 95% confidence interval = 1.03–2.30; P = 0.037). The association with prevalent diabetes was replicated among European myocardial infarction survivors (rate ratio = 2.09; 95% confidence interval = 1.17–3.76; P = 0.013).

Conclusions: These results suggest that genetic variation in the IL-1 gene family is associated with measures of glucose homeostasis and prevalent diabetes.

Genetic variation in the IL-1 gene family is associated with measures of glucose homeostasis and prevalent diabetes.
Ethnic Variation in Fat and Lean Body Mass and the Association with Insulin Resistance
Authors: Lear Scott A. , Kohli Simi , Bondy Gregory P. , Tchernof André , Sniderman Allan D.
Published: October 2009

Context: Body fat distribution varies among different ethnic groups, yet less is known regarding differences in lean mass and how this may affect insulin resistance.

Objective: Our objective was to compare total body fat to lean mass ratio (F:LM) in Aboriginal, Chinese, European, and South Asian individuals with differences in insulin resistance.

Participants, Design, and Setting: Aboriginal (196), Chinese (222), European (202), and South Asian (208) individuals were recruited across a range of body mass index to participate in this cross-sectional community study.

Main Outcome Measures: Total body fat, lean mass, and insulin resistance were assessed using homeostasis model assessment (HOMA).

Results: After adjustment for confounders and at a given body fat, South Asian men had less lean mass than Aboriginal [3.42 kg less; 95% confidence interval (CI) = 1.55–5.29], Chinese (3.01 kg less; 95% CI = 1.33–4.70), and European (3.57 kg less; 95% CI = 1.82–5.33) men, whereas South Asian women had less lean mass than Aboriginal (1.98 kg less; 95% CI = 0.45–3.50), Chinese (2.24 kg less; 95% CI = 0.81–3.68), and European (2.97 kg less; 95% CI = 1.67–4.27) women. In adjusted models, F:LM was higher in South Asian compared with Chinese and European men and higher in South Asian compared with Aboriginal, Chinese, and European women (P < 0.01 for all). Insulin and HOMA were greatest in South Asians after adjustment; however, these differences were no longer apparent when F:LM was considered.

Conclusions: South Asians have a phenotype of high fat mass and low lean mass, which may account for greater levels of insulin and HOMA compared with other ethnic groups.
Obese Children with Low Birth Weight Demonstrate Impaired β-Cell Function during Oral Glucose Tolerance Test
Authors: Brufani Claudia , Grossi Armando , Fintini Danilo , Tozzi Alberto , Nocerino Valentina , Patera Patrizia Ippolita , Ubertini Graziamaria , Porzio Ottavia , Barbetti Fabrizio , Cappa Marco
Published: October 2009

Objective: Epidemiological studies have shown an association between birth weight and future risk of type 2 diabetes, with individuals born either small or large for gestational age at increased risk. We sought to investigate the influence of birth weight on the relation between insulin sensitivity and β-cell function in obese children.

Subjects and Methods: A total of 257 obese/overweight children (mean body mass index-sd score, 2.2 ± 0.3), aged 11.6 ± 2.3 yr were divided into three groups according to birth weight percentile: 44 were small for gestational age (SGA), 161 were appropriate for gestational age (AGA), and 52 were large for gestational age (LGA). Participants underwent a 3-h oral glucose tolerance test with glucose, insulin, and C-peptide measurements. Homeostasis model of assessment for insulin resistance, insulinogenic index, and disposition index were calculated to evaluate insulin sensitivity and β-cell function. Glucose and insulin area under the curve (AUC) were also considered. One-way ANOVA was used to compare the three groups.

Results: SGA and LGA subjects had higher homeostasis model of assessment for insulin resistance than AGA subjects, but they diverged when oral glucose tolerance test response was considered. Indeed, SGA subjects showed higher glucose AUC and lower insulinogenic and disposition indexes. Insulin AUC was not different between groups, but when singular time points were considered, SGA subjects had lower insulin levels at 30 min and higher insulin levels at 180 min.

Conclusions: SGA obese children fail to adequately compensate for their reduced insulin sensitivity, manifesting deficit in early insulin response and reduced disposition index that results in higher glucose AUC. Thus, SGA obese children show adverse metabolic outcomes compared to AGAs and LGAs.

Small for gestational age obese children manifest a deficit in the early insulin response that results in a higher glucose area under the curve.
MicroRNA 132 Regulates Nutritional Stress-Induced Chemokine Production through Repression of SirT1
Authors: Strum Jay C. , Johnson Jennifer H. , Ward James , Xie Hongbo , Feild John , Hester Austin , Alford Alexander , Waters K. Michelle
Published: October 2009

Human adipose tissue secretes a number of proinflammatory mediators that may contribute to the pathophysiology of obesity-related disorders. Understanding the regulatory pathways that control their production is paramount to developing effective therapeutics to treat these diseases. Using primary human adipose-derived stem cells as a source of preadipocytes and in vitro differentiated adipocytes, we found IL-8 and monocyte chemoattractant protein-1 (MCP-1) are constitutively secreted by both cell types and induced in response to serum deprivation. MicroRNA profiling revealed the rapid induction of microRNA 132 (miR-132) in these cells when switched to serum-free medium. Furthermore, miR-132 overexpression was sufficient to induce nuclear factor-κB translocation, acetylation of p65, and production of IL-8 and MCP-1. Inhibitors of miR-132 decreased acetylated p65 and partially inhibited the production of IL-8 and MCP-1 induced by serum deprivation. MiR-132 was shown to inhibit silent information regulator 1 (SirT1) expression through a miR-132 binding site in the 3′-untranslated region of SirT1. Thus, in response to nutritional availability, induction of miR-132 decreases SirT1-mediated deacetylation of p65 leading to activation of nuclear factor-κB and transcription of IL-8 and MCP-1 in primary human preadipocytes and in vitro differentiated adipocytes.