Publications by authors named "Robert Augustin"

26 Publications

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Challenges in tackling energy expenditure as obesity therapy: From preclinical models to clinical application.

Mol Metab 2021 Apr 18;51:101237. Epub 2021 Apr 18.

Cardio Metabolic Diseases Research, Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany. Electronic address:

Background: A chronic imbalance of energy intake and energy expenditure results in excess fat storage. The obesity often caused by this overweight is detrimental to the health of millions of people. Understanding both sides of the energy balance equation and their counter-regulatory mechanisms is critical to the development of effective therapies to treat this epidemic.

Scope Of Review: Behaviors surrounding ingestion have been reviewed extensively. This review focuses more specifically on energy expenditure regarding bodyweight control, with a particular emphasis on the organs and attractive metabolic processes known to reduce bodyweight. Moreover, previous and current attempts at anti-obesity strategies focusing on energy expenditure are highlighted. Precise measurements of energy expenditure, which consist of cellular, animal, and human models, as well as measurements of their translatability, are required to provide the most effective therapies.

Major Conclusions: A precise understanding of the components surrounding energy expenditure, including tailored approaches based on genetic, biomarker, or physical characteristics, must be integrated into future anti-obesity treatments. Further comprehensive investigations are required to define suitable treatments, especially because the complex nature of the human perspective remains poorly understood.
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http://dx.doi.org/10.1016/j.molmet.2021.101237DOI Listing
April 2021

The glucose-dependent insulinotropic polypeptide (GIP) regulates body weight and food intake via CNS-GIPR signaling.

Cell Metab 2021 Apr 10;33(4):833-844.e5. Epub 2021 Feb 10.

Institute for Diabetes and Obesity, Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany; German Center for Diabetes Research (DZD), Neuherberg, Germany.

Uncertainty exists as to whether the glucose-dependent insulinotropic polypeptide receptor (GIPR) should be activated or inhibited for the treatment of obesity. Gipr was recently demonstrated in hypothalamic feeding centers, but the physiological relevance of CNS Gipr remains unknown. Here we show that HFD-fed CNS-Gipr KO mice and humanized (h)GIPR knockin mice with CNS-hGIPR deletion show decreased body weight and improved glucose metabolism. In DIO mice, acute central and peripheral administration of acyl-GIP increases cFos neuronal activity in hypothalamic feeding centers, and this coincides with decreased body weight and food intake and improved glucose handling. Chronic central and peripheral administration of acyl-GIP lowers body weight and food intake in wild-type mice, but shows blunted/absent efficacy in CNS-Gipr KO mice. Also, the superior metabolic effect of GLP-1/GIP co-agonism relative to GLP-1 is extinguished in CNS-Gipr KO mice. Our data hence establish a key role of CNS Gipr for control of energy metabolism.
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http://dx.doi.org/10.1016/j.cmet.2021.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035082PMC
April 2021

SGLT2 is not expressed in pancreatic α- and β-cells, and its inhibition does not directly affect glucagon and insulin secretion in rodents and humans.

Mol Metab 2020 12 5;42:101071. Epub 2020 Sep 5.

Pole of Endocrinology, Diabetes, and Nutrition (EDIN), Institute of Experimental and Clinical Research (IREC), Université Catholique de Louvain (UCLouvain), 1200, Brussels, Belgium. Electronic address:

Objective: Sodium-glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i), or gliflozins, are anti-diabetic drugs that lower glycemia by promoting glucosuria, but they also stimulate endogenous glucose and ketone body production. The likely causes of these metabolic responses are increased blood glucagon levels, and decreased blood insulin levels, but the mechanisms involved are hotly debated. This study verified whether or not SGLT2i affect glucagon and insulin secretion by a direct action on islet cells in three species, using multiple approaches.

Methods: We tested the in vivo effects of two selective SGLT2i (dapagliflozin, empagliflozin) and a SGLT1/2i (sotagliflozin) on various biological parameters (glucosuria, glycemia, glucagonemia, insulinemia) in mice. mRNA expression of SGLT2 and other glucose transporters was assessed in rat, mouse, and human FACS-purified α- and β-cells, and by analysis of two human islet cell transcriptomic datasets. Immunodetection of SGLT2 in pancreatic tissues was performed with a validated antibody. The effects of dapagliflozin, empagliflozin, and sotagliflozin on glucagon and insulin secretion were assessed using isolated rat, mouse and human islets and the in situ perfused mouse pancreas. Finally, we tested the long-term effect of SGLT2i on glucagon gene expression.

Results: SGLT2 inhibition in mice increased the plasma glucagon/insulin ratio in the fasted state, an effect correlated with a decline in glycemia. Gene expression analyses and immunodetections showed no SGLT2 mRNA or protein expression in rodent and human islet cells, but moderate SGLT1 mRNA expression in human α-cells. However, functional experiments on rat, mouse, and human (29 donors) islets and the in situ perfused mouse pancreas did not identify any direct effect of dapagliflozin, empagliflozin or sotagliflozin on glucagon and insulin secretion. SGLT2i did not affect glucagon gene expression in rat and human islets.

Conclusions: The data indicate that the SGLT2i-induced increase of the plasma glucagon/insulin ratio in vivo does not result from a direct action of the gliflozins on islet cells.
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http://dx.doi.org/10.1016/j.molmet.2020.101071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554656PMC
December 2020

Glucose-dependent insulinotropic polypeptide (GIP) and cardiovascular disease.

Peptides 2020 03 2;125:170174. Epub 2019 Nov 2.

Center for Clinical Metabolic Research, Gentofte Hospital, University of Copenhagen, Hellerup, Denmark; Steno Diabetes Center Copenhagen, Gentofte, Denmark; Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Accumulating evidence suggests that glucose-dependent insulinotropic polypeptide (GIP) in addition to its involvement in type 2 diabetic pathophysiology may be involved in the development of obesity and the pathogenesis of cardiovascular disease. In this review, we outline recent preclinical and clinical cardiovascular-related discoveries about GIP. These include chronotropic and blood pressure-lowering effects of GIP. Furthermore, GIP has been suggested to control vasodilation via secretion of nitric oxide, and vascular leukocyte adhesion and inflammation via expression and secretion of endothelin 1. Also, GIP seems to regulate circulating lipids via effects on adipose tissue uptake and metabolism of lipids. Lastly, we discuss how dysmetabolic conditions such as obesity and type 2 diabetes may shift the actions of GIP in an atherogenic direction, and we provide a perspective on the therapeutic potential of GIP receptor agonism and antagonism in cardiovascular diseases. We conclude that GIP actions may have implications for the development of cardiovascular disease, but also that the potential of GIP-based drugs for the treatment of cardiovascular disease currently is uncertain.
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http://dx.doi.org/10.1016/j.peptides.2019.170174DOI Listing
March 2020

The role of GIP and pancreatic GLP-1 in the glucoregulatory effect of DPP-4 inhibition in mice.

Diabetologia 2019 10 14;62(10):1928-1937. Epub 2019 Aug 14.

Department of Surgery, University of Michigan, 2800 Plymouth Road, Ann Arbor, MI, 48109, USA.

Aims/hypothesis: Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are two peptides that function to promote insulin secretion. Dipeptidyl peptidase-4 (DPP-4) inhibitors increase the bioavailability of both GLP-1 and GIP but the dogma continues to be that it is the increase in GLP-1 that contributes to the improved glucose homeostasis. We have previously demonstrated that pancreatic rather than intestinal GLP-1 is necessary for improvements in glucose homeostasis in mice. Therefore, we hypothesise that a combination of pancreatic GLP-1 and GIP is necessary for the full effect of DPP-4 inhibitors on glucose homeostasis.

Methods: We have genetically engineered mouse lines in which the preproglucagon gene (Gcg) is absent in the entire body (GcgRA) or is expressed exclusively in the intestine (GcgRA) or pancreas and duodenum (GcgRA). These mice were used to examine oral glucose tolerance and GLP-1 and GIP responses to a DPP-4 inhibitor alone, or in combination with incretin receptor antagonists.

Results: Administration of the DPP-4 inhibitor, linagliptin, improved glucose tolerance in GcgRA mice and control littermates and in GcgRA and GcgRA mice. The potent GLP-1 receptor antagonist, exendin-[9-39] (Ex9), blunted improvements in glucose tolerance in linagliptin-treated control mice and in GcgRA mice. Ex9 had no effect on glucose tolerance in linagliptin-treated GcgRA or in GcgRA mice. In addition to GLP-1, linagliptin also increased postprandial plasma levels of GIP to a similar degree in all genotypes. When linagliptin was co-administered with a GIP-antagonising antibody, the impact of linagliptin was partially blunted in wild-type mice and was fully blocked in GcgRA mice.

Conclusions/interpretation: Taken together, these data suggest that increases in pancreatic GLP-1 and GIP are necessary for the full effect of DPP-4 inhibitors on glucose tolerance.
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http://dx.doi.org/10.1007/s00125-019-4963-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732043PMC
October 2019

The GLP-1 Analogs Liraglutide and Semaglutide Reduce Atherosclerosis in ApoE and LDLr Mice by a Mechanism That Includes Inflammatory Pathways.

JACC Basic Transl Sci 2018 Dec 21;3(6):844-857. Epub 2018 Nov 21.

Global Research, Novo Nordisk A/S, Maaloev, Denmark.

The glucagon-like peptide-1 receptor agonists (GLP-1RAs) liraglutide and semaglutide reduce cardiovascular risk in type 2 diabetes patients. The mode of action is suggested to occur through modified atherosclerotic progression. In this study, both of the compounds significantly attenuated plaque lesion development in apolipoprotein E-deficient (ApoE) mice and low-density lipoprotein receptor-deficient (LDLr) mice. This attenuation was partly independent of weight and cholesterol lowering. In aortic tissue, exposure to a Western diet alters expression of genes in pathways relevant to the pathogenesis of atherosclerosis, including leukocyte recruitment, leukocyte rolling, adhesion/extravasation, cholesterol metabolism, lipid-mediated signaling, extracellular matrix protein turnover, and plaque hemorrhage. Treatment with semaglutide significantly reversed these changes. These data suggest GLP-1RAs affect atherosclerosis through an anti-inflammatory mechanism.
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http://dx.doi.org/10.1016/j.jacbts.2018.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314963PMC
December 2018

Generation of a highly diverse panel of antagonistic chicken monoclonal antibodies against the GIP receptor.

MAbs 2017 04 5;9(3):536-549. Epub 2017 Jan 5.

b Crystal Bioscience , Emeryville , CA , USA.

Raising functional antibodies against G protein-coupled receptors (GPCRs) is challenging due to their low density expression, instability in the absence of the cell membrane's lipid bilayer and frequently short extracellular domains that can serve as antigens. In addition, a particular therapeutic concept may require an antibody to not just bind the receptor, but also act as a functional receptor agonist or antagonist. Antagonizing the glucose-dependent insulinotropic polypeptide (GIP) receptor may open up new therapeutic modalities in the treatment of diabetes and obesity. As such, a panel of monoclonal antagonistic antibodies would be a useful tool for in vitro and in vivo proof of concept studies. The receptor is highly conserved between rodents and humans, which has contributed to previous mouse and rat immunization campaigns generating very few usable antibodies. Switching the immunization host to chicken, which is phylogenetically distant from mammals, enabled the generation of a large and diverse panel of monoclonal antibodies containing 172 unique sequences. Three-quarters of all chicken-derived antibodies were functional antagonists, exhibited high-affinities to the receptor extracellular domain and sampled a broad epitope repertoire. For difficult targets, including GPCRs such as GIPR, chickens are emerging as valuable immunization hosts for therapeutic antibody discovery.
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http://dx.doi.org/10.1080/19420862.2016.1276683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384726PMC
April 2017

TUSC5 regulates insulin-mediated adipose tissue glucose uptake by modulation of GLUT4 recycling.

Mol Metab 2015 Nov 28;4(11):795-810. Epub 2015 Aug 28.

Institute of Food, Nutrition and Health, ETH Zurich, Schwerzenbach, Switzerland.

Objective: Failure to properly dispose of glucose in response to insulin is a serious health problem, occurring during obesity and is associated with type 2 diabetes development. Insulin-stimulated glucose uptake is facilitated by the translocation and plasma membrane fusion of vesicles containing glucose transporter 4 (GLUT4), the rate-limiting step of post-prandial glucose disposal.

Methods: We analyzed the role of Tusc5 in the regulation of insulin-stimulated Glut4-mediated glucose uptake in vitro and in vivo. Furthermore, we measured Tusc5 expression in two patient cohorts.

Results: Herein, we report that TUSC5 controls insulin-stimulated glucose uptake in adipocytes, in vitro and in vivo. TUSC5 facilitates the proper recycling of GLUT4 and other key trafficking proteins during prolonged insulin stimulation, thereby enabling proper protein localization and complete vesicle formation, processes that ultimately enable insulin-stimulated glucose uptake. Tusc5 knockout mice exhibit impaired glucose disposal and TUSC5 expression is predictive of glucose tolerance in obese individuals, independent of body weight. Furthermore, we show that TUSC5 is a PPARγ target and in its absence the anti-diabetic effects of TZDs are significantly blunted.

Conclusions: Collectively, these findings establish TUSC5 as an adipose tissue-specific protein that enables proper protein recycling, linking the ubiquitous vesicle traffic machinery with tissue-specific insulin-mediated glucose uptake into adipose tissue and the maintenance of a healthy metabolic phenotype in mice and humans.
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http://dx.doi.org/10.1016/j.molmet.2015.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632119PMC
November 2015

The apical sorting signal for human GLUT9b resides in the N-terminus.

Mol Cell Biochem 2013 Apr 30;376(1-2):163-73. Epub 2013 Jan 30.

Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO 63110, USA.

The two splice variants of human glucose transporter 9 (hGLUT9) are targeted to different polarized membranes. hGLUT9a traffics to the basolateral membrane, whereas hGLUT9b traffics to the apical region. This study examines the sorting mechanism of these variants, which differ only in their N-terminal domain. Mutating a di-leucine motif unique to GLUT9a did not affect targeting. Chimeric proteins were made using GLUT1, a basolaterally targeted transporter, and GLUT3, an apically targeted protein whose signal lies in the C-terminus. Overexpression of the chimeric proteins in polarized cells demonstrates that the N-terminus of hGLUT9b contains a signal capable of redirecting GLUT1 to the apical membrane. The N-terminus of hGLUT9a, however, does not contain a basolateral signal sufficient enough to redirect GLUT3. Portions of the GLUT9a N-terminus were substituted with corresponding portions of the GLUT9b N-terminus to determine the motif responsible for apical targeting. The first 16 amino acids were not found to be a sufficient apical signal. The last ten amino acids of the N-termini differ only in amino-acid class at one location. In the B-form, leucine, a hydrophobic residue, is substituted for lysine, a basic residue, found in the A-form. However, mutation of the leucine in hGLUT9b to a lysine resulted in retention of the apical signal. We therefore believe the apical signal exists as an interplay between the final ten amino acids of the N-terminus and another motif within the protein such as the intracellular loop or other motifs within the N-terminus.
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http://dx.doi.org/10.1007/s11010-013-1564-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5028210PMC
April 2013

GTPase ARFRP1 is essential for normal hepatic glycogen storage and insulin-like growth factor 1 secretion.

Mol Cell Biol 2012 Nov 27;32(21):4363-74. Epub 2012 Aug 27.

Department of Experimental Diabetology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany.

The GTPase ADP-ribosylation factor-related protein 1 (ARFRP1) is located at the trans-Golgi compartment and regulates the recruitment of Arf-like 1 (ARL1) and its effector golgin-245 to this compartment. Here, we show that liver-specific knockout of Arfrp1 in the mouse (Arfrp1(liv-/-)) resulted in early growth retardation, which was associated with reduced hepatic insulin-like growth factor 1 (IGF1) secretion. Accordingly, suppression of Arfrp1 in primary hepatocytes resulted in a significant reduction of IGF1 release. However, the hepatic secretion of IGF-binding protein 2 (IGFBP2) was not affected in the absence of ARFRP1. In addition, Arfrp1(liv-/-) mice exhibited decreased glucose transport into the liver, leading to a 50% reduction of glycogen stores as well as a marked retardation of glycogen storage after fasting and refeeding. These abnormalities in glucose metabolism were attributable to reduced protein levels and intracellular retention of the glucose transporter GLUT2 in Arfrp1(liv-/-) livers. As a consequence of impaired glucose uptake into the liver, the expression levels of carbohydrate response element binding protein (ChREBP), a transcription factor regulated by glucose concentration, and its target genes (glucokinase and pyruvate kinase) were markedly reduced. Our data indicate that ARFRP1 in the liver is involved in the regulation of IGF1 secretion and GLUT2 sorting and is thereby essential for normal growth and glycogen storage.
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http://dx.doi.org/10.1128/MCB.00522-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486143PMC
November 2012

Functional characterisation of human SGLT-5 as a novel kidney-specific sodium-dependent sugar transporter.

FEBS Lett 2012 Feb 30;586(3):248-53. Epub 2011 Dec 30.

Department of CardioMetabolic Diseases Research, Boehringer Ingelheim Pharma GmbH & Co.KG, Biberach, Germany.

Sodium glucose cotransporters (SGLT) actively catalyse carbohydrate transport across cellular membranes. Six of the 12 known SGLT family members have the capacity to bind and/or transport monosaccharides (SGLT-1 to 6); of these, all but SGLT-5 have been characterised. Here we demonstrate that human SGLT-5 is exclusively expressed in the kidney. Four splice variants were detected and the most abundant SGLT-5-mRNA was functionally characterised. SGLT-5 mediates sodium-dependent [(14)C]-α-methyl-D-glucose (AMG) transport that can be inhibited by mannose, fructose, glucose, and galactose. Uptake studies using demonstrated high capacity transport for mannose and fructose and, to a lesser extent, glucose, AMG, and galactose. SGLT-5 mediated mannose, fructose and AMG transport was weakly (μM potency) inhibited by SGLT-2 inhibitors. In summary, we have characterised SGLT-5 as a kidney mannose transporter. Further studies are warranted to explore the physiological role of SGLT-5.
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http://dx.doi.org/10.1016/j.febslet.2011.12.027DOI Listing
February 2012

Identification of a potential biomarker for FABP4 inhibition: the power of lipidomics in preclinical drug testing.

J Biomol Screen 2011 Jun 4;16(5):467-75. Epub 2011 May 4.

1Institute for Bioinformatics and Systems Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany.

The fatty acid binding protein 4 (FABP4) belongs to the family of lipid chaperones that control intracellular fluxes and compartmentalization of their respective ligands (e.g., fatty acids). FABP4, which is almost exclusively expressed in adipocytes and macrophages, contributes to the development of insulin resistance and atherosclerosis in mice. Lack of FABP4 protects against the development of insulin resistance associated with genetic or diet-induced obesity in mice. Furthermore, total or macrophage-specific FABP4 deficiency is protective against atherosclerosis in apolipoprotein E-deficient mice. The FABP4 small-molecule inhibitor BMS309403 has demonstrated efficacy in mouse models for type 2 diabetes mellitus and atherosclerosis, resembling phenotypes of mice with FABP4 deficiency. However, despite the therapeutically attractive long-term effects of FABP4 inhibition, an acute biomarker for drug action is lacking. The authors applied mass spectrometry lipidomics analysis to in vitro and in vivo (plasma and adipose tissue) samples upon inhibitor treatment. They report the identification of a potential biomarker for acute in vivo FABP4 inhibition that is applicable for further investigations and can be implemented in simple and fast-flow injection mass spectrometry assays. In addition, this approach can be considered a proof-of-principle study that can be applied to other lipid-pathway targeting mechanisms.
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http://dx.doi.org/10.1177/1087057111402200DOI Listing
June 2011

The protein family of glucose transport facilitators: It's not only about glucose after all.

Authors:
Robert Augustin

IUBMB Life 2010 May;62(5):315-33

Department of Cardiometabolic Diseases Research, Boehringer-Ingelheim Pharma GmbH&Co KG, Biberach a.d. Riss, Germany.

The protein family of facilitative glucose transporters comprises 14 isoforms that share common structural features such as 12 transmembrane domains, N- and C-termini facing the cytoplasm of the cell, and a N-glycosylation side either within the first or fifth extracellular loop. Based on their sequence homology, three classes can be distinguished: class I includes GLUT1-4 and GLUT14, class II the "odd transporters" GLUT5, 7, 9, 11, and class III the "even transporters" GLUT6, 8, 10, 12 and the proton driven myoinositol transporter HMIT (or GLUT13). With the cloning and characterization of the more recent class II and III isoforms, it became apparent that despite their structural similarities, the different isoforms not only show a distinct tissue-specific expression pattern but also show distinct characteristics such as alternative splicing, specific (sub)cellular localization, and affinities for a spectrum of substrates. This review summarizes the current understanding of the physiological role for the various transport facilitators based on human genetically inherited disorders or single-nucleotide polymorphisms and knockout mice models. The emphasis of the review will be on the potential functional role of the more recent isoforms.
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http://dx.doi.org/10.1002/iub.315DOI Listing
May 2010

Lysosomal localization of GLUT8 in the testis--the EXXXLL motif of GLUT8 is sufficient for its intracellular sorting via AP1- and AP2-mediated interaction.

FEBS J 2009 Jul 11;276(14):3729-43. Epub 2009 Jun 11.

Institute of Chemistry and Biochemistry, Department of Membrane Biochemistry, Freie Universität & Charité Universitätsmedizin Berlin, Takustrasse 6, Berlin, Germany.

The class III sugar transport facilitator GLUT8 co-localizes with the lysosomal protein LAMP1 in heterologous expression systems. GLUT8 carries a [D/E]XXXL[L/I]-type dileucine sorting signal that has been postulated to retain the protein in an endosomal/lysosomal compartment via interactions with clathrin adaptor protein (AP) complexes. However, contradictory findings have been described regarding the subcellular localization of the endogenous GLUT8 and the adaptor proteins that interact with its dileucine motif. Here we demonstrate that endogenous GLUT8 is localized in a late endosomal/lysosomal compartment of spermatocytes and spermatids, and that the adaptor complexes AP1 and AP2, but not AP3 or AP4, interact with its N-terminal intracellular domain (NICD). In addition, fusion of the GLUT8 NICD to the tailless lumenal domain of the IL-2 receptor alpha chain (TAC) protein (interleukin-2 receptor a chain) targeted the protein to intracellular membranes, indicating that its N-terminal dileucine signal is sufficient for endosomal/lysosomal targeting of the transporter. The localization and targeting of GLUT8 show striking similarities to sorting mechanisms reported for lysosomal proteins. Therefore, we suggest a potential role for GLUT8 in the so far unexplored substrate transport across intracellular membranes.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07089.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2730553PMC
July 2009

Neuronal functions, feeding behavior, and energy balance in Slc2a3+/- mice.

Am J Physiol Endocrinol Metab 2008 Nov 9;295(5):E1084-94. Epub 2008 Sep 9.

Department of Pharmacology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany.

Homozygous deletion of the gene of the neuronal glucose transporter GLUT3 (Slc2a3) in mice results in embryonic lethality, whereas heterozygotes (Slc2a3+/-) are viable. Here, we describe the characterization of heterozygous mice with regard to neuronal function, glucose homeostasis, and, since GLUT3 might be a component of the neuronal glucose-sensing mechanism, food intake and energy balance. Levels of GLUT3 mRNA and protein in brain were reduced by 50% in Slc2a3+/- mice. Electrographic features examined by electroencephalographic recordings give evidence for slightly but significantly enhanced cerebrocortical activity in Slc2a3+/- mice. In addition, Slc2a3+/- mice were slightly more sensitive to an acoustic startle stimulus (elevated startle amplitude and reduced prepulse inhibition). However, systemic behavioral testing revealed no other functional abnormalities, e.g., in coordination, reflexes, motor abilities, anxiety, learning, and memory. Furthermore, no differences in body weight, blood glucose, and insulin levels were detected between wild-type and Slc2a3+/- littermates. Food intake as monitored randomly or after intracerebroventricular administration of 2-deoxyglucose or d-glucose, or food choice for carbohydrates/fat was not affected in Slc2a3+/- mice. Taken together, our data indicate that, in contrast to Slc2a1, a single allele of Slc2a3 is sufficient for maintenance of neuronal energy supply, motor abilities, learning and memory, and feeding behavior.
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http://dx.doi.org/10.1152/ajpendo.90491.2008DOI Listing
November 2008

ADP-ribosylation factor-like GTPase ARFRP1 is required for trans-Golgi to plasma membrane trafficking of E-cadherin.

J Biol Chem 2008 Oct 28;283(40):27179-88. Epub 2008 Jul 28.

Department of Pharmacology, German Institute of Human Nutrition Potsdam-Rehbruecke, 14558 Nuthetal, Germany.

ADP-ribosylation factor-related protein 1 (ARFRP1) plays a specific role in Golgi function controlling recruitment of GRIP domain proteins and ARL1 to the trans-Golgi. Deletion of the mouse Arfrp1 gene causes embryonic lethality during early gastrulation, because epiblast cells detach from the ectodermal cell layer and do not differentiate to mesodermal tissue. Here we show that in Arfrp1(-/-) embryos E-cadherin is mistargeted to intracellular compartments, whereas in control embryos it is present at the cell surface of trophectodermal and ectodermal cells. In enterocytes of intestine-specific Arfrp1 null mutants (Arfrp1(vil)(-/-)), E-cadherin is associated with intracellular membranes, partially colocalizing with the cis-Golgi marker GM130 or with punctae close to the cell surface. In contrast, in control enterocytes E-cadherin is exclusively located in the lateral membranes. In addition, ARL1 is dislocated from Golgi membranes to the cytosol of Arfrp1(vil)(-/-) enterocytes. Depletion of endogenous ARFRP1 by RNA interference leads to a dislocation of E-cadherin from the cell surface in HeLa cells and to a reduced cell aggregation in Ltk(-)Ecad cells. ARFRP1 was coimmunoprecipitated in a complex with E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, and p120(ctn) from lysates of Madin-Darby canine kidney cells stably expressing myc-ARFRP1. These data indicate that knock-out of Arfrp1 disrupts the trafficking of E-cadherin through the Golgi and suggest an essential role of the GTPase in trans-Golgi network function.
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http://dx.doi.org/10.1074/jbc.M802108200DOI Listing
October 2008

Targeted disruption of Slc2a8 (GLUT8) reduces motility and mitochondrial potential of spermatozoa.

Mol Membr Biol 2008 Apr;25(3):224-35

Department of Pharmacology, German Institute of Human Nutrition Potsdam-Rehbruecke, Germany.

GLUT8 is a class 3 sugar transport facilitator which is predominantly expressed in testis and also detected in brain, heart, skeletal muscle, adipose tissue, adrenal gland, and liver. Since its physiological function in these tissues is unknown, we generated a Slc2a8 null mouse and characterized its phenotype. Slc2a8 knockout mice appeared healthy and exhibited normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role for maintenance of whole body glucose homeostasis. However, analysis of the offspring distribution of heterozygous mating indicated a lower number of Slc2a8 knockout offspring (30.5:47.3:22.1%, Slc2a8(+/+), Slc2a8(+/-), and Slc2a8(-/-) mice, respectively) resulting in a deviation (p=0.0024) from the expected Mendelian distribution. This difference was associated with lower ATP levels, a reduced mitochondrial membrane potential and a significant reduction of sperm motility of the Slc2a8 knockout in comparison to wild-type spermatozoa. In contrast, number and survival rate of spermatozoa were not altered. These data indicate that GLUT8 plays an important role in the energy metabolism of sperm cells.
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http://dx.doi.org/10.1080/09687680701855405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2557070PMC
April 2008

Uncoupling protein 1 expression in murine skeletal muscle increases AMPK activation, glucose turnover, and insulin sensitivity in vivo.

Physiol Genomics 2008 May 18;33(3):333-40. Epub 2008 Mar 18.

Department of Pharmacology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany.

Uncoupling of oxidative phosphorylation represents a potential target for the treatment of hyperglycemia and insulin resistance in obesity and type 2 diabetes. The present study investigated whether the expression of uncoupling protein 1 in skeletal muscles of transgenic (mUCP1 TG) mice modulates insulin action in major insulin target tissues in vivo. Euglycemic-hyperinsulinemic clamps (17 pM x kg lean body mass(-1) x min(-1)) were performed in 9-mo-old hemizygous male mUCP1 TG mice and wild-type (WT) littermates matched for body composition. mUCP1 TG mice exhibited fasting hypoglycemia and hypoinsulinemia compared with WT mice, whereas fasting hepatic glucose production rates were comparable in both genotypes. mUCP1 TG mice were markedly more sensitive to insulin action compared with WT mice and displayed threefold higher glucose infusion rates, enhanced skeletal muscle and white adipose tissue glucose uptake, and whole body glycolysis rates. In the absence of alterations in plasma adiponectin concentrations, acceleration of insulin-stimulated glucose turnover in skeletal muscle of mUCP1 TG mice was accompanied by increased phosphorylated Akt-to-Akt and phosphorylated AMP-activated protein kinase (AMPK)-to-AMPK ratios compared with WT mice. UCP1-mediated uncoupling of oxidative phosphorylation in skeletal muscle was paralleled by AMPK activation and thereby stimulated insulin-mediated glucose uptake in skeletal muscle.
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http://dx.doi.org/10.1152/physiolgenomics.00226.2007DOI Listing
May 2008

A highly conserved hydrophobic motif in the exofacial vestibule of fructose transporting SLC2A proteins acts as a critical determinant of their substrate selectivity.

Mol Membr Biol 2007 Sep-Dec;24(5-6):455-63

Department of Physiology, University of Alberta, Edmonton, Alberta, Canada.

The substrate specificity of the facilitated hexose transporter, GLUT, family, (gene SLC2A) is highly varied. Some appear to be able to translocate both glucose and fructose, while the ability to handle 2-deoxyglucose and galactose does not necessarily correlate with the other two hexoses. It has become generally accepted that a central substrate binding/translocation site determines which hexoses can be transported. However, a recent study showed that a single point mutation of a hydrophobic residue in GLUTs 2, 5 & 7 removed their ability to transport fructose without affecting the kinetics of glucose permeation. This residue is in the 7th transmembrane helix, facing the aqueous pore and lies close to the opening of the exofacial vestibule. This study expands these observations to include the other class II GLUTs (9 & 11) and shows that a three amino acid motif (NXI/NXV) appears to be critical in determining if fructose can access the translocation mechanism. GLUT11 can also transport fructose, but it has the motif DSV at the same position, which appears to function in the same manner as NXI and when all three residues are replaced with NAV fructose transport lost. These results are discussed in relation to possible roles for hydrophobic residues lining the aqueous pore at the opening of the exofacial vestibule. Finally, the possibility that the translocation binding site may not be the sole determinant of substrate specificity for these proteins is examined.
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http://dx.doi.org/10.1080/09687680701298143DOI Listing
November 2007

Utilization of DR1 as true RARE in regulating the Ssm, a novel retinoic acid-target gene in the mouse testis.

J Endocrinol 2007 Mar;192(3):539-51

Department of Obstetrics and Gynecology, Washington University School of Medicine, 660 S Euclid Avenue, St Louis, Missouri 63110, USA.

Various nuclear receptors form dimers to activate target genes via specific response elements located within promoters or enhancers. Retinoid X receptor (RXR) serves as a dimerization partner for many nuclear receptors including retinoic acid receptor (RAR) and peroxisome proliferator-activated receptor (PPAR). Dimers show differential preference towards directly repeated response elements with 1-5 nucleotide spacing, and direct repeat 1 (DR1) is a promiscuous element which recruits RAR/RXR, RXR/RXR, and PPAR/RXR in vitro. In the present investigation, we report identification of a novel RAR/RXR target gene which is regulated by DR1s in the promoter region. This gene, namely spermatocyte-specific marker (Ssm), recruits all the three combinations of nuclear receptors in vitro, but in vivo regulation is observed by trans-retinoic acid-activated RAR/RXR dimer. Indeed, chromatin immunoprecipitation experiment demonstrates binding of RARbeta and RXRalpha in the promoter region of the Ssm. Interestingly, expression of Ssm is almost exclusively observed in spermatocytes in the adult mouse testis, where RA signaling is known to regulate developmental program of male germ cells. The results show that Ssm is a RAR/RXR target gene uniquely using DR1 and exhibits stage-specific expression in the mouse testis with potential function in later stages of spermatogenesis. This finding exemplifies usage of DR1s as retinoic acid response element (RARE) under a specific in vivo context.
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http://dx.doi.org/10.1677/JOE-06-0115DOI Listing
March 2007

Ablation of the cholesterol transporter adenosine triphosphate-binding cassette transporter G1 reduces adipose cell size and protects against diet-induced obesity.

Endocrinology 2007 Apr 28;148(4):1561-73. Epub 2006 Dec 28.

Department of Pharmacology, German Institute of Human Nutrition, Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, D-14558 Nuthetal, Germany.

The ATP-binding cassette transporter G1 (ABCG1) catalyzes export of cellular cholesterol from macrophages and hepatocytes. Here we identify an additional function of ABCG1 in the regulation of adiposity in screens of the Drosophila melanogaster and the New Zealand obese (NZO) mouse genomes. Insertion of modified transposable elements of the P-family upstream of CG17646, the Drosophila ortholog of Abcg1, generated lines of flies with increased triglyceride stores. In NZO mice, an Abcg1 variant was identified in a suggestive adiposity quantitative trait locus and was associated with higher expression of the gene in white adipose tissue. Targeted disruption of Abcg1 in mice resulted in reduced body weight gain (8.42+/-0.6 g in Abcg1-/- vs. 13.07+/-1.1 g in Abcg1+/+ mice) and adipose tissue mass gain (3.78+/-1.3 g in Abcg1-/- vs. 9.39+/-1.6 g in Abcg1+/+ mice) detected over a period of 12 wk. The reduction of adipose tissue mass in Abcg1-/- mice was associated with markedly decreased size of the adipocytes. In contrast to their wild-type littermates, male Abcg1-/- mice exhibited no high-fat diet-induced impairment of glucose tolerance and fatty liver. Furthermore, Abcg1-/- mice possess decreased food intake and elevated total energy expenditure (Abcg1-/- mice, 748.1+/-5.4 kJ/kg metabolic body mass; Abcg1+/+ mice, 684.3+/-5.0 kJ/kg metabolic body mass; P=0.011), body temperature (Abcg1-/- mice, 37.82+/-0.29 C; Abcg1+/+ mice, 36.83+/-0.24 C; P<0.05), and locomotor activity (Abcg1-/- mice, 3655+/-189 counts/12 h during dark phase; Abcg1+/+ mice, 2445+/-235 counts/12 h during dark phase; P<0.01). Our data indicate a previously unrecognized role of ABCG1 in the regulation of energy balance and triglyceride storage.
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http://dx.doi.org/10.1210/en.2006-1244DOI Listing
April 2007

Mouse glucose transporter 9 splice variants are expressed in adult liver and kidney and are up-regulated in diabetes.

Mol Endocrinol 2006 Mar 17;20(3):686-97. Epub 2005 Nov 17.

Washington University School of Medicine, Department of Obstetrics/Gynelcology, Box 8064, St. Louis, Missouri 63110, USA.

A novel glucose transporter (GLUT), mouse GLUT9 (mGLUT9), was recently cloned from mouse 7-d embryonic cDNA. Several splice variants of mGLUT9 were described, two of which were cloned (mGLUT9a and mGLUT9a Delta 209-316). This study describes the cloning and characterization of another splice variant, mGLUT9b. Cloned from adult liver, mGLUT9b is identical to mGLUT9a except at the amino terminus. Based on analysis of the genomic structure, the different amino termini result from alternative transcriptional/translational start sites. Expression and localization of these two mGLUT9 splice variants were examined in control and diabetic adult mouse tissues and in cell lines. RT-PCR analysis demonstrated expression of mGLUT9a in several tissues whereas mGLUT9b was observed primarily in liver and kidney. Using a mGLUT9-specific antibody, Western blot analysis of total membrane fractions from liver and kidney detected a single, wide band, migrating at approximately 55 kDa. This band shifted to a lower molecular mass when deglycosylated with peptide-N-glycosidase F. Both forms were present in liver and kidney. Immunohistochemical localization demonstrated basolateral distribution of mGLUT9 in liver hepatocytes and the expression of mGLUT9 in specific tubules in the outer cortex of the kidney. To investigate the alternative amino termini, mGLUT9a and mGLUT9b were overexpressed in kidney epithelium cell lines. Subcellular fractions localized both forms to the plasma membrane. Immunofluorescent staining of polarized Madin Darby canine kidney cells overexpressing mGLUT9 depicted a basolateral distribution for both splice variants. Finally, mGLUT9 protein expression was significantly increased in the kidney and liver from streptozotocin-induced diabetic mice compared with nondiabetic animals.
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http://dx.doi.org/10.1210/me.2005-0010DOI Listing
March 2006

GLUT8 contains a [DE]XXXL[LI] sorting motif and localizes to a late endosomal/lysosomal compartment.

Traffic 2005 Dec;6(12):1196-212

Institute for Human Nutrition, Department of Pharmacology, 14482 Potsdam-Rehbrücke, Germany.

Glucose transporter 8 (GLUT8) contains a cytoplasmic N-terminal dileucine motif and localizes to a thus far unidentified intracellular compartment. Translocation of GLUT8 to the plasma membrane (PM) was found in insulin-treated mouse blastocysts. Using overexpression of GLUT8 in adipocytes and neuronal cells however, insulin treatment or depolarization of the cells did not lead to GLUT8 PM translocation in other studies. In addition, other experiments showing dynamin-dependent endocytosis of GLUT8 suggested that GLUT8 recycles between an endosomal compartment and the PM. To reveal the functional/physiological role of GLUT8, we studied its subcellular localization in 3T3L1, HEK293 and CHO cells. We show that GLUT8 does not co-localize with GLUT4 and does not redistribute to the PM after treatment with insulin, ionophores or okadaic acid in these cell lines. Once endocytosed, GLUT8 does not recycle to the PM. GLUT8 localizes to late endosomes and lysosomes. An interspecies GLUT8 - sequence alignment revealed the presence of a highly conserved late endosomal/lysosomal-targeting motif ([DE]XXXL[LI]). Changing the glutamate to arginine as found in GLUT4 (RRXXXLL) alters GLUT8 endocytosis and retains the transporter at the PM. Furthermore, sorting GLUT8 to late endosomes/lysosomes does not require prior presence of GLUT8 at the PM followed by its endocytosis. In summary, GLUT8 does not reside in a recycling vesicle pool and is distinct from GLUT4. From our data, we postulate a role for GLUT8 in transport of hexoses across intracellular membranes, for example in specific compartments of GLUT8 expression such as the acrosome of mature spermatozoa or secretory granules in neurons. Furthermore, a role for GLUT8 in hexose transport across the lysosomal membrane, a transport mechanism that has long been suggested but unexplained, is discussed.
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http://dx.doi.org/10.1111/j.1600-0854.2005.00354.xDOI Listing
December 2005

Identification and characterization of human glucose transporter-like protein-9 (GLUT9): alternative splicing alters trafficking.

J Biol Chem 2004 Apr 22;279(16):16229-36. Epub 2004 Jan 22.

Department of Obstetrics/Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

The recently cloned human GLUT9 gene, which maps to chromosome 4p15.3-p16, consists of 12 exons coding for a 540-amino acid protein. Based on a sequence entry (NCBI accession number BC018897) and screening of expressed sequence tags, we have cloned an alternative splice variant of GLUT9 from human kidney cDNA. The RNA of this splice variant consists of 13 exons and codes for a putative protein of 512 amino acids (GLUT9DeltaN). The predicted proteins differ only in their N terminus, suggesting a different subcellular localization and possible physiological role. Screening human tissue RNA by reverse transcription-PCR showed that GLUT9 is expressed mainly in kidney, liver, placenta, and leukocytes, whereas GLUT9DeltaN was detected only in kidney and placenta. The GLUT9 protein localized by immunohistochemistry to human kidney proximal tubules, and subcellular fractionation of human kidney revealed the GLUT9 protein in plasma membranes and high density microsomal membranes. Treatment of kidney membrane proteins with peptide N-glycosidase F showed that GLUT9 and GLUT9DeltaN are expressed in vivo. Localization of GLUT9 and GLUT9DeltaN in three kidney-derived cell lines revealed a plasma membrane distribution for GLUT9 in COS-7 and HEK293 cells, whereas GLUT9DeltaN showed a perinuclear pattern and plasma membrane staining in COS-7 and HEK293 cells, respectively. In polarized Madin-Darby canine kidney cells, GLUT9 trafficked to the basolateral membrane, whereas GLUT9DeltaN localized to the apical membrane. Using heterologous expression of GLUT9 in Xenopus oocytes, GLUT9 appears to be a functional isoform with low affinity for deoxyglucose. Deoxyglucose transport mediated by GLUT9 was not inhibited by cytochalasin B. GLUT9 did not bind cytochalasin B as shown by a cytochalasin B binding assay, indicating a similar behavior of GLUT9 compared with GLUT5.
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http://dx.doi.org/10.1074/jbc.M312226200DOI Listing
April 2004

Constitutive expression of CYP1A1 in bovine cumulus oocyte-complexes in vitro: mechanisms and biological implications.

Endocrinology 2004 Apr 15;145(4):1594-601. Epub 2004 Jan 15.

Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle, Saale, Germany.

The arylhydrocarbon receptor (AhR) is known to mediate toxic responses to dioxin (2,3,7,8-tetrachlorodibenzo-p- dioxin) and related compounds and has been extensively characterized from a toxicological viewpoint. However, it has recently been reported that the AhR may have a central role in ovarian physiology. To investigate the role of AhR during oocyte maturation, we analyzed the expression of AhR, its nuclear partner AhR nuclear translocator, and the major target gene CYP1A1, in bovine cumulus-oocyte complexes (COCs) by semiquantitative RT-PCR and Western blot. Coexpression of AhR and AhR nuclear translocator was observed in both oocytes and surrounding cumulus cells before and after in vitro maturation (IVM). Furthermore, after IVM, both cell types showed a clear up-regulation of AhR mRNA compared with the expression at 0 h. Constitutive expression of CYP1A1 mRNA was observed in immature oocytes at the background level, whereas no expression was observed in the surrounding cumulus cells. Interestingly, a significant increase in CYP1A1 expression level was observed in both oocytes and cumulus cells after IVM. To further investigate the role of AhR in CYP1A1 up-regulation and oocyte maturation, COCs were treated throughout IVM with the AhR antagonists, alpha-naphthoflavone and resveratrol. Both antagonists decreased the level of CYP1A1 in COCs compared with controls. Furthermore, CYP1A1 down-regulation was accompanied by a reduced ability of oocytes to complete in vitro maturation until metaphase II stage. These results suggest that CYP1A1 induction in COCs is necessary for correct proceeding of in vitro oocyte maturation in bovine and suggest a physiological role of AhR during resumption of meiosis.
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http://dx.doi.org/10.1210/en.2003-1254DOI Listing
April 2004

Toxic effects of in vitro exposure to p-tert-octylphenol on bovine oocyte maturation and developmental competence.

Biol Reprod 2003 Aug 16;69(2):462-8. Epub 2003 Apr 16.

Department of Anatomy and Cell Biology, Faculty of Medicine, Martin Luther University Halle/Wittenberg, D-06097 Halle (Saale), Germany.

Alkylphenolic compounds are a widespread family of xenoestrogens. High concentrations of these substances are present in sewage sludge that is spread on arable land and pasture as fertilizer. Because of their known endocrine system-disrupting activity, alkylphenols represent a potential risk for the reproductive health of farm animals. In this study, the impact of p-tert-octylphenol (OP) on the developmental competence of bovine oocytes was evaluated. Endocrine activity of OP was investigated for its effect on estrogen receptors alpha and beta (ERalpha and ERbeta) and progesterone receptor (PR) mRNA levels. Cumulus-oocyte complexes (COCs) were exposed during in vitro maturation to serial concentrations of OP (1-0.0001 microg/ml) and were compared with vehicle-treated controls and a group of COCs treated with 17 beta-estradiol (E2). A dose-related decrease in the percentage of oocytes that completed maturation after 24 h and in oocyte fertilization competence was observed at doses of OP as low as 0.01 microg/ml. Groups treated with > or =0.001 microg/ml OP showed impaired embryo development. No adverse effects of E2 were observed. In the E2-treated COCs, ERalpha mRNA was decreased but PR mRNA was upregulated compared with controls. Treatment with 0.001 and 0.0001 microg/ml OP induced a decrease in ERalpha mRNA, but ERbeta and PR mRNA were not affected. Treatment with 0.01 microg/ml OP did not produce changes in the expression of any of the mRNAs studied. OP impairs meiotic progression and developmental competence of bovine oocytes without demonstrating clear estrogen-mimic activity.
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http://dx.doi.org/10.1095/biolreprod.102.010355DOI Listing
August 2003