The Na ؉ /I ؊ symporter (NIS) is a key membrane glycoprotein that mediates active I ؊ transport i... more The Na ؉ /I ؊ symporter (NIS) is a key membrane glycoprotein that mediates active I ؊ transport in the thyroid and other tissues. Upon isolation of the cDNA encoding NIS, 10 NIS mutations that cause congenital iodide transport defect have been identified. Three of these mutations (T354P, G395R, and Q267E) have been thoroughly characterized at the molecular level. All three NIS mutant proteins are correctly targeted to the plasma membrane; however, whereas Q267E displays minimal activity, T354P and G395R are inactive. Here, we show that in contrast to these mutants, G543E NIS matures only partially and is retained intracellularly; thus, it is not targeted properly to the cell surface, apparently because of faulty folding. These findings indicate that the G543 residue plays significant roles in NIS maturation and trafficking. Remarkably, NIS activity was rescued by small neutral amino acid substitutions (volume < 129 Å 3 ) at this position, suggesting that G543 is in a tightly packed region of NIS. (Molecular
The uptake of tobramycin was measured in Escherichia coli membrane vesicles prepared in KMES [K(+... more The uptake of tobramycin was measured in Escherichia coli membrane vesicles prepared in KMES [K(+)-2-(N-morpholino)ethanesulfonic acid] buffer at pH 6.6. Uptake occurred in vesicles energized with ascorbic acid and phenazine methosulfate, in which the electrical potential (delta psi) was -120 mV, but not in vesicles energized with D-lactate (delta psi = -95 mV). The addition of nigericin to vesicles energized with D-lactate did not induce tobramycin uptake despite an increase in delta psi to -110 mV. However, when delta psi was increased or decreased by the addition of nigericin or valinomycin, respectively, uptake in vesicles energized with ascorbic acid and phenazine methosulfate was stimulated or inhibited, respectively, confirming studies with whole cells showing that uptake of aminoglycosides is gated by delta psi rather than by proton motive force (delta microH+) or delta pH. N-ethylmaleimide prevented uptake, suggesting that the aminoglycoside transporter is a cytoplasmic mem...
The Na ؉ /I ؊ symporter (NIS), a 618-amino acid membrane glycoprotein that catalyzes the active a... more The Na ؉ /I ؊ symporter (NIS), a 618-amino acid membrane glycoprotein that catalyzes the active accumulation of I ؊ into thyroid cells, was identified and characterized at the molecular level in our laboratory (Dai, G., Levy, O., and Carrasco, N. (1996) Nature 379, 458-460). Because mature NIS is highly glycosylated, it migrates in SDS-polyacrylamide gel electrophoresis as a broad polypeptide of higher molecular mass (ϳ90-110 kDa) than nonglycosylated NIS (ϳ50 kDa). Using site-directed mutagenesis, we substituted both separately and simultaneously the asparagine residues in all three putative N-linked glycosylation consensus sequences of NIS with glutamine and assessed the effects of the mutations on function and stability of NIS in COS cells. All mutants were active and displayed 50-90% of wild-type NIS activity, including the completely nonglycosylated triple mutant. This demonstrates that to a considerable extent, function and stability of NIS are preserved in the partial or even total absence of N-linked glycosylation. We also found that Asn 225 is glycosylated, thus proving that the hydrophilic loop that contains this amino acid residue faces the extracellular milieu rather than the cytosol as previously suggested. We demonstrated that the NH 2 terminus faces extracellularly as well. A new secondary structure model consistent with these findings is proposed.
The sodium/iodide symporter (NIS) is the essential plasma membrane protein that mediates active i... more The sodium/iodide symporter (NIS) is the essential plasma membrane protein that mediates active iodide (I-) transport into the thyroid gland, the first step in the biosynthesis of the thyroid hormones—the master regulators of intermediary metabolism. NIS couples the inward translocation of I- against its electrochemical gradient to the inward transport of Na+ down its electrochemical gradient. For nearly 50 years before its molecular identification, NIS was already the molecule at the center of the single most effective internal radiation cancer therapy ever devised: radioiodide (131I-) treatment for thyroid cancer. Mutations in NIS cause congenital hypothyroidism, which must be treated immediately after birth to prevent stunted growth and cognitive deficiency. To date, the structure of NIS has been unknown. Here, we report three structures of rat NIS, determined by single-particle cryo-electron microscopy (cryo-EM): one with no substrates bound, one with 2 Na+ and 1 I- bound, and o...
The Journal of Clinical Endocrinology & Metabolism, 2021
Context Iodide transport defect (ITD) (Online Mendelian Inheritance in Man No. 274400) is an unco... more Context Iodide transport defect (ITD) (Online Mendelian Inheritance in Man No. 274400) is an uncommon cause of dyshormonogenic congenital hypothyroidism due to loss-of-function variants in the SLC5A5 gene, which encodes the sodium/iodide symporter (NIS), causing deficient iodide accumulation in thyroid follicular cells. Objective This work aims to determine the molecular basis of a patient’s ITD clinical phenotype. Methods The propositus was diagnosed with dyshormonogenic congenital hypothyroidism with minimal 99mTc-pertechnetate accumulation in a eutopic thyroid gland. The propositus SLC5A5 gene was sequenced. Functional in vitro characterization of the novel NIS variant was performed. Results Sanger sequencing revealed a novel homozygous missense p.G561E NIS variant. Mechanistically, the G561E substitution reduces iodide uptake, because targeting of G561E NIS to the plasma membrane is reduced. Biochemical analyses revealed that G561E impairs the recognition of an adjacent tryptoph...
Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 2004
UNLABELLED Pertechnetate (as (99m)TcO(4)(-)), (123)I(-), and (131)I(-) have a long and successful... more UNLABELLED Pertechnetate (as (99m)TcO(4)(-)), (123)I(-), and (131)I(-) have a long and successful history of use in the diagnosis and therapy of thyroid cancer, with uptake into thyroid tissue mediated by the sodium-iodide symporter (NIS). NIS has also emerged as a potential target for radiotherapy of nonthyroid malignancies that express the endogenous or transfected symporter. Perrhenates (as (188)ReO(4)(-) and (186)ReO(4)(-)) are promising therapeutic substrates of NIS, although less is known about their behavior in vivo. In this study, we endeavored to characterize the biologic behavior of perrhenate, especially in relation to iodide and pertechnetate, to better explore its possible therapeutic role. METHODS We describe the simultaneous biodistribution and uptake in vivo of iodide, pertechnetate, and perrhenate in groups of healthy CD1 mice, either with or without coadministration of perchlorate (ClO(4)(-)), a potent NIS inhibitor. Animals administered single radiopharmaceuticals...
7 Viktoriya Paroder, Juan P. Nicola, Christopher S. Ginter, 8 and Nancy Carrasco 9 10 a Departmen... more 7 Viktoriya Paroder, Juan P. Nicola, Christopher S. Ginter, 8 and Nancy Carrasco 9 10 a Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461. 11 b Department of Cellular and Molecular Physiology, Yale University School of Medicine, New 12 Haven, CT 06510. 13 14 Present addresses: 1 Department of Radiology, Montefiore Medical Center. Bronx, NY 10467. 2 15 Department of Physiology and Biophysics, Weill Cornell Medical School, New York, NY 10065. 16 # These authors contributed equally to this work. 17 * To whom correspondence should be addressed. Department of Cellular and Molecular Physiology, 18 Yale University School of Medicine. 333 Cedar Street. New Haven, CT 06510. Phone: (203) 78519 4117. E-mail: [email protected]. 20 21 Abbreviated title: R124H NIS is intrinsically active. 22
Background: We propose micro single-photon emission computed tomography/computed tomography imagi... more Background: We propose micro single-photon emission computed tomography/computed tomography imaging of the hNIS (human sodium/iodide symporter) to noninvasively quantify adeno-associated virus 9 (AAV9)-mediated gene expression in a murine model of peripheral artery disease. Methods: AAV9-hNIS (2×10 11 viral genome particles) was injected into nonischemic or ischemic gastrocnemius muscles of C57Bl/6J mice following unilateral hindlimb ischemia ± the α-sialidase NA (neuraminidase). Control nonischemic limbs were injected with phosphate buffered saline or remained noninjected. Twelve mice underwent micro single-photon emission computed tomography/computed tomography imaging after serial injection of pertechnetate ( 99m TcO 4 − ), a NIS substrate, up to 28 days after AAV9-hNIS injection. Twenty four animals were euthanized at selected times over 1 month for ex vivo validation. Forty-two animals were imaged with 99m TcO 4 − ± the selective NIS inhibitor perchlorate on day 10, to ascertai...
Proceedings of the National Academy of Sciences of the United States of America, Oct 24, 2017
Hypothyroidism, a metabolic disease characterized by low thyroid hormone (TH) and high thyroid-st... more Hypothyroidism, a metabolic disease characterized by low thyroid hormone (TH) and high thyroid-stimulating hormone (TSH) levels in the serum, is strongly associated with nonalcoholic fatty liver disease (NAFLD). Hypothyroidism-induced NAFLD has generally been attributed to reduced TH signaling in the liver with a consequent decrease in lipid utilization. Here, we found that mildly hypothyroid mice develop NAFLD without down-regulation of hepatic TH signaling or decreased hepatic lipid utilization. NAFLD was induced by impaired suppression of adipose tissue lipolysis due to decreased insulin secretion and to a reduced response of adipose tissue itself to insulin. This condition leads to increased shuttling of fatty acids (FAs) to the liver, where they are esterified and accumulated as triglycerides. Lipid accumulation in the liver induces hepatic insulin resistance, which leads to impaired suppression of endogenous glucose production after feeding. Hepatic insulin resistance, synergi...
The sodium/iodide symporter (NIS) mediates active iodide (I −) accumulation in the thyroid, the f... more The sodium/iodide symporter (NIS) mediates active iodide (I −) accumulation in the thyroid, the first step in thyroid hormone (TH) biosynthesis. Mutations in the SLC5A5 gene encoding NIS that result in a non-functional protein lead to congenital hypothyroidism due to I − transport defect (ITD). ITD is a rare autosomal disorder that, if not treated promptly in infancy, can cause mental retardation, as the TH decrease results in improper development of the nervous system. However, in some patients, hypothyroidism has been ameliorated by unusually large amounts of dietary I −. Here we report the first NIS knockout (KO) mouse model, obtained by targeting exons 6 and 7 of the Slc5a5 gene. In NIS KO mice, in the thyroid, stomach, and salivary gland, NIS is absent, and hence there is no active accumulation of the NIS substrate pertechnetate (99m TcO 4 −). NIS KO mice showed undetectable serum T 4 and very low serum T 3 levels when fed a diet supplying the minimum I − requirement for rodents. These hypothyroid mice displayed oxidative stress in the thyroid, but not in the brown adipose tissue or liver. Feeding the mice a high-I − diet partially rescued TH biosynthesis, demonstrating that, at high I − concentrations, I − enters the thyroid through routes other than NIS. Iodine is an essential constituent of the thyroid hormones (THs), which are phenolic rings joined by an ether link and iodinated at either three positions (3,5,3′-tri-iodo-l-thyronine, or T 3) or four positions (3,5,3′,5′-tetra-i odo-l-thyronine, or T 4). The THs are required for the proper development of the central nervous system 1 , skeleton 2 , and lungs 3 in the fetus and the newborn and for intermediary metabolism at all ages 4. Therefore, both hyper and hypothyroidism are diseased states with serious systemic ramifications. Iodide (I −) is an extremely scarce nutrient in the environment and is supplied only in the diet. The Na + /I − symporter (NIS), a member of solute carrier family 5 (SLC5), is the plasma membrane protein that actively transports I − into the thyroid follicular cells, using as its driving force the Na + gradient generated by the Na + /K + ATPase. NIS transports I − electrogenically with a 2 Na + : 1 I − stoichiometry 1. Since our group cloned the cDNA coding for NIS 5 , we have reported in vivo and in vitro that NIS is regulated transcriptionally and post-transcriptionally by thyroid stimulating hormone (TSH) 6-9. We have identified residues that coordinate NIS substrates 10-12 , and elucidated the basis for the efficient transport of I − by NIS at the physiological (submicromolar) concentrations of I − in the plasma 13. Besides the thyroid gland, NIS is functionally expressed in other tissues including lactating breast 14 , breast cancer 9, 14 , salivary glands 14 , and stomach 1, 9, 14. NIS-mediated I − transport is the key first step in TH biosynthesis, which occurs partly intracellularly and partly in the colloid, an extracellular compartment. I − reaches the colloid through an I − efflux process that has not been fully elucidated. The genetic characterization of patients affected by Pendred syndrome, a disorder characterized by goiter and a partial I − organification defect 15 , led to the identification of pendrin (SLC26A4) as a protein that may mediate I − efflux into the colloid. However, pendrin knockout (KO) mice do not display hypothyroidism 16 , indicating that other mechanisms are also involved in thyroid apical I − efflux. The Na + /monocarboxylate transporter (SMCT) (Slc5a8) is expressed at the apical membrane of thyroid follicular cells 17. SMCT was
The rat thyroid Na ؉ /I ؊ symporter (NIS) was expressed in Xenopus laevis oocytes and characteriz... more The rat thyroid Na ؉ /I ؊ symporter (NIS) was expressed in Xenopus laevis oocytes and characterized using electrophysiological, tracer uptake, and electron microscopic methods. NIS activity was found to be electrogenic and Na ؉-dependent (Na ؉ > > Li ؉ > > H ؉). The apparent affinity constants for Na ؉ and I ؊ were 28 ؎ 3 mM and 33 ؎ 9 M, respectively. Stoichiometry of Na ؉ / anion cotransport was 2:1. NIS was capable of transporting a wide variety of anions (I
BACKGROUND & AIMS: Caudal-related homeodomain transcription factors CDX1 and CDX2 regulate gut de... more BACKGROUND & AIMS: Caudal-related homeodomain transcription factors CDX1 and CDX2 regulate gut development and differentiation of intestinal epithelial cells; they are candidate tumor suppressors of colorectal carcinomas. Because the functions of CDX1 and CDX2 in the colonic epithelium are not fully understood, we sought to identify genes that they target. METHODS: We conducted a chromatin immunoprecipitation (ChIP) screen to identify genes that bind the CDX transcription factors. Expression of target genes was analyzed in colon cells and tissues from Cdx1 Ϫ/Ϫ , Cdx2 ϩ/Ϫ , Apc ϩ/⌬716 , and wild-type (control) mice. RESULTS: Using the ChIP screen, we identified solute carrier family 5, member 8 (SLC5A8, also known as SMCT1) as a direct target of CDX1 and CDX2. CDX transcription factors bind to the promoter region of SLC5A8 and transactivate SLC5A8 reporter constructs. Overexpression of Cdx1 or Cdx2 in human colon cancer cell lines induced expression of endogenous SLC5A8, whereas CDX1 and CDX2 knockdowns reduced its level. Consistently, Slc5a8 expression was significantly reduced in colons of Cdx1 Ϫ/Ϫ or Cdx2 ϩ/Ϫ mice compared with wild-type mice. Slc5a8 levels were also reduced in colonic adenomatous polyps and hamartomas from Apc ϩ/⌬716 and Cdx2 ϩ/Ϫ mutant mice, respectively, compared with adjacent normal colon tissues. CONCLUSIONS: CDX1 and CDX2 bind the promoter region of SLC5A8 and up-regulate its expression in cultured cells and in colonic epithelium. SLC5A8 transports monocarboxylates such as pyruvate, lactate, and butyrate; CDX1 and CDX2 might therefore regulate the uptake of these substances in the colon. C DX genes are human orthologues of Drosophila caudal 1 and code for homeodomain transcription factors. 2 Three CDX family members, CDX1, CDX2, and CDX4, are expressed at early embryonic stages and play important roles in placentation, axis elongation, and hematopoiesis. 3-6 Cdx1 Ϫ/Ϫ mice and Cdx2 ϩ/Ϫ mice suffer homeotic shifts in the skeleton, 3,4,7 whereas Cdx2 Ϫ/Ϫ mouse embryos do not survive beyond 3.5-5.5 days postcoitum, because of abnormal placenta caused by developmental disorders in the trophectoderm.
The rat thyroid Na+/I- symporter (NIS) was expressed in Xenopus laevis oocytes and characterized ... more The rat thyroid Na+/I- symporter (NIS) was expressed in Xenopus laevis oocytes and characterized using electrophysiological, tracer uptake, and electron microscopic methods. NIS activity was found to be electrogenic and Na+-dependent (Na+ > Li+ > H+). The apparent affinity constants for Na+ and I- were 28 +/- 3 mM and 33 +/- 9 microM, respectively. Stoichiometry of Na+/anion cotransport was 2:1. NIS was capable of transporting a wide variety of anions (I-, ClO3-, SCN-, SeCN-, NO3-, Br-, BF4-, IO4-, BrO3-, but perchlorate (ClO4-) was not transported. In the absence of anion substrate, NIS exhibited a Na+-dependent leak current (approximately 35% of maximum substrate-induced current) with an apparent Na+ affinity of 74 +/- 14 mM and a Hill coefficient (n) of 1. In response to step voltage changes, NIS exhibited current transients that relaxed with a time constant of 8-14 ms. Presteady-state charge movements (integral of the current transients) versus voltage relations obey a Bol...
Iodide is an important regulator of thyroid activity. Its excess elicits the Wolff-Chaikoff effec... more Iodide is an important regulator of thyroid activity. Its excess elicits the Wolff-Chaikoff effect, characterized by an acute suppression of thyroid hormone synthesis, which has been ascribed to serum TSH reduction or TGF-β increase and production of iodolipids in the thyroid. These alterations take hours/days to occur, contrasting with the promptness of Wolff-Chaikoff effect. We investigated whether acute iodide administration could trigger events that precede those changes, such as reduction of sodium-iodide symporter (NIS) mRNA abundance and adenylation, and if perchlorate treatment could counteract them. Rats subjected or not to methylmercaptoimidazole treatment (0.03%) received NaI (2,000 μg/0.5 ml saline) or saline intraperitoneally and were killed 30 min up to 24 h later. Another set of animals was treated with iodide and perchlorate, in equimolar doses. NIS mRNA content was evaluated by Northern blotting and real-time PCR, and NIS mRNA poly(A) tail length by rapid amplificat...
The Na ؉ /I ؊ symporter (NIS) is a key membrane glycoprotein that mediates active I ؊ transport i... more The Na ؉ /I ؊ symporter (NIS) is a key membrane glycoprotein that mediates active I ؊ transport in the thyroid and other tissues. Upon isolation of the cDNA encoding NIS, 10 NIS mutations that cause congenital iodide transport defect have been identified. Three of these mutations (T354P, G395R, and Q267E) have been thoroughly characterized at the molecular level. All three NIS mutant proteins are correctly targeted to the plasma membrane; however, whereas Q267E displays minimal activity, T354P and G395R are inactive. Here, we show that in contrast to these mutants, G543E NIS matures only partially and is retained intracellularly; thus, it is not targeted properly to the cell surface, apparently because of faulty folding. These findings indicate that the G543 residue plays significant roles in NIS maturation and trafficking. Remarkably, NIS activity was rescued by small neutral amino acid substitutions (volume < 129 Å 3 ) at this position, suggesting that G543 is in a tightly packed region of NIS. (Molecular
The uptake of tobramycin was measured in Escherichia coli membrane vesicles prepared in KMES [K(+... more The uptake of tobramycin was measured in Escherichia coli membrane vesicles prepared in KMES [K(+)-2-(N-morpholino)ethanesulfonic acid] buffer at pH 6.6. Uptake occurred in vesicles energized with ascorbic acid and phenazine methosulfate, in which the electrical potential (delta psi) was -120 mV, but not in vesicles energized with D-lactate (delta psi = -95 mV). The addition of nigericin to vesicles energized with D-lactate did not induce tobramycin uptake despite an increase in delta psi to -110 mV. However, when delta psi was increased or decreased by the addition of nigericin or valinomycin, respectively, uptake in vesicles energized with ascorbic acid and phenazine methosulfate was stimulated or inhibited, respectively, confirming studies with whole cells showing that uptake of aminoglycosides is gated by delta psi rather than by proton motive force (delta microH+) or delta pH. N-ethylmaleimide prevented uptake, suggesting that the aminoglycoside transporter is a cytoplasmic mem...
The Na ؉ /I ؊ symporter (NIS), a 618-amino acid membrane glycoprotein that catalyzes the active a... more The Na ؉ /I ؊ symporter (NIS), a 618-amino acid membrane glycoprotein that catalyzes the active accumulation of I ؊ into thyroid cells, was identified and characterized at the molecular level in our laboratory (Dai, G., Levy, O., and Carrasco, N. (1996) Nature 379, 458-460). Because mature NIS is highly glycosylated, it migrates in SDS-polyacrylamide gel electrophoresis as a broad polypeptide of higher molecular mass (ϳ90-110 kDa) than nonglycosylated NIS (ϳ50 kDa). Using site-directed mutagenesis, we substituted both separately and simultaneously the asparagine residues in all three putative N-linked glycosylation consensus sequences of NIS with glutamine and assessed the effects of the mutations on function and stability of NIS in COS cells. All mutants were active and displayed 50-90% of wild-type NIS activity, including the completely nonglycosylated triple mutant. This demonstrates that to a considerable extent, function and stability of NIS are preserved in the partial or even total absence of N-linked glycosylation. We also found that Asn 225 is glycosylated, thus proving that the hydrophilic loop that contains this amino acid residue faces the extracellular milieu rather than the cytosol as previously suggested. We demonstrated that the NH 2 terminus faces extracellularly as well. A new secondary structure model consistent with these findings is proposed.
The sodium/iodide symporter (NIS) is the essential plasma membrane protein that mediates active i... more The sodium/iodide symporter (NIS) is the essential plasma membrane protein that mediates active iodide (I-) transport into the thyroid gland, the first step in the biosynthesis of the thyroid hormones—the master regulators of intermediary metabolism. NIS couples the inward translocation of I- against its electrochemical gradient to the inward transport of Na+ down its electrochemical gradient. For nearly 50 years before its molecular identification, NIS was already the molecule at the center of the single most effective internal radiation cancer therapy ever devised: radioiodide (131I-) treatment for thyroid cancer. Mutations in NIS cause congenital hypothyroidism, which must be treated immediately after birth to prevent stunted growth and cognitive deficiency. To date, the structure of NIS has been unknown. Here, we report three structures of rat NIS, determined by single-particle cryo-electron microscopy (cryo-EM): one with no substrates bound, one with 2 Na+ and 1 I- bound, and o...
The Journal of Clinical Endocrinology & Metabolism, 2021
Context Iodide transport defect (ITD) (Online Mendelian Inheritance in Man No. 274400) is an unco... more Context Iodide transport defect (ITD) (Online Mendelian Inheritance in Man No. 274400) is an uncommon cause of dyshormonogenic congenital hypothyroidism due to loss-of-function variants in the SLC5A5 gene, which encodes the sodium/iodide symporter (NIS), causing deficient iodide accumulation in thyroid follicular cells. Objective This work aims to determine the molecular basis of a patient’s ITD clinical phenotype. Methods The propositus was diagnosed with dyshormonogenic congenital hypothyroidism with minimal 99mTc-pertechnetate accumulation in a eutopic thyroid gland. The propositus SLC5A5 gene was sequenced. Functional in vitro characterization of the novel NIS variant was performed. Results Sanger sequencing revealed a novel homozygous missense p.G561E NIS variant. Mechanistically, the G561E substitution reduces iodide uptake, because targeting of G561E NIS to the plasma membrane is reduced. Biochemical analyses revealed that G561E impairs the recognition of an adjacent tryptoph...
Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 2004
UNLABELLED Pertechnetate (as (99m)TcO(4)(-)), (123)I(-), and (131)I(-) have a long and successful... more UNLABELLED Pertechnetate (as (99m)TcO(4)(-)), (123)I(-), and (131)I(-) have a long and successful history of use in the diagnosis and therapy of thyroid cancer, with uptake into thyroid tissue mediated by the sodium-iodide symporter (NIS). NIS has also emerged as a potential target for radiotherapy of nonthyroid malignancies that express the endogenous or transfected symporter. Perrhenates (as (188)ReO(4)(-) and (186)ReO(4)(-)) are promising therapeutic substrates of NIS, although less is known about their behavior in vivo. In this study, we endeavored to characterize the biologic behavior of perrhenate, especially in relation to iodide and pertechnetate, to better explore its possible therapeutic role. METHODS We describe the simultaneous biodistribution and uptake in vivo of iodide, pertechnetate, and perrhenate in groups of healthy CD1 mice, either with or without coadministration of perchlorate (ClO(4)(-)), a potent NIS inhibitor. Animals administered single radiopharmaceuticals...
7 Viktoriya Paroder, Juan P. Nicola, Christopher S. Ginter, 8 and Nancy Carrasco 9 10 a Departmen... more 7 Viktoriya Paroder, Juan P. Nicola, Christopher S. Ginter, 8 and Nancy Carrasco 9 10 a Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461. 11 b Department of Cellular and Molecular Physiology, Yale University School of Medicine, New 12 Haven, CT 06510. 13 14 Present addresses: 1 Department of Radiology, Montefiore Medical Center. Bronx, NY 10467. 2 15 Department of Physiology and Biophysics, Weill Cornell Medical School, New York, NY 10065. 16 # These authors contributed equally to this work. 17 * To whom correspondence should be addressed. Department of Cellular and Molecular Physiology, 18 Yale University School of Medicine. 333 Cedar Street. New Haven, CT 06510. Phone: (203) 78519 4117. E-mail: [email protected]. 20 21 Abbreviated title: R124H NIS is intrinsically active. 22
Background: We propose micro single-photon emission computed tomography/computed tomography imagi... more Background: We propose micro single-photon emission computed tomography/computed tomography imaging of the hNIS (human sodium/iodide symporter) to noninvasively quantify adeno-associated virus 9 (AAV9)-mediated gene expression in a murine model of peripheral artery disease. Methods: AAV9-hNIS (2×10 11 viral genome particles) was injected into nonischemic or ischemic gastrocnemius muscles of C57Bl/6J mice following unilateral hindlimb ischemia ± the α-sialidase NA (neuraminidase). Control nonischemic limbs were injected with phosphate buffered saline or remained noninjected. Twelve mice underwent micro single-photon emission computed tomography/computed tomography imaging after serial injection of pertechnetate ( 99m TcO 4 − ), a NIS substrate, up to 28 days after AAV9-hNIS injection. Twenty four animals were euthanized at selected times over 1 month for ex vivo validation. Forty-two animals were imaged with 99m TcO 4 − ± the selective NIS inhibitor perchlorate on day 10, to ascertai...
Proceedings of the National Academy of Sciences of the United States of America, Oct 24, 2017
Hypothyroidism, a metabolic disease characterized by low thyroid hormone (TH) and high thyroid-st... more Hypothyroidism, a metabolic disease characterized by low thyroid hormone (TH) and high thyroid-stimulating hormone (TSH) levels in the serum, is strongly associated with nonalcoholic fatty liver disease (NAFLD). Hypothyroidism-induced NAFLD has generally been attributed to reduced TH signaling in the liver with a consequent decrease in lipid utilization. Here, we found that mildly hypothyroid mice develop NAFLD without down-regulation of hepatic TH signaling or decreased hepatic lipid utilization. NAFLD was induced by impaired suppression of adipose tissue lipolysis due to decreased insulin secretion and to a reduced response of adipose tissue itself to insulin. This condition leads to increased shuttling of fatty acids (FAs) to the liver, where they are esterified and accumulated as triglycerides. Lipid accumulation in the liver induces hepatic insulin resistance, which leads to impaired suppression of endogenous glucose production after feeding. Hepatic insulin resistance, synergi...
The sodium/iodide symporter (NIS) mediates active iodide (I −) accumulation in the thyroid, the f... more The sodium/iodide symporter (NIS) mediates active iodide (I −) accumulation in the thyroid, the first step in thyroid hormone (TH) biosynthesis. Mutations in the SLC5A5 gene encoding NIS that result in a non-functional protein lead to congenital hypothyroidism due to I − transport defect (ITD). ITD is a rare autosomal disorder that, if not treated promptly in infancy, can cause mental retardation, as the TH decrease results in improper development of the nervous system. However, in some patients, hypothyroidism has been ameliorated by unusually large amounts of dietary I −. Here we report the first NIS knockout (KO) mouse model, obtained by targeting exons 6 and 7 of the Slc5a5 gene. In NIS KO mice, in the thyroid, stomach, and salivary gland, NIS is absent, and hence there is no active accumulation of the NIS substrate pertechnetate (99m TcO 4 −). NIS KO mice showed undetectable serum T 4 and very low serum T 3 levels when fed a diet supplying the minimum I − requirement for rodents. These hypothyroid mice displayed oxidative stress in the thyroid, but not in the brown adipose tissue or liver. Feeding the mice a high-I − diet partially rescued TH biosynthesis, demonstrating that, at high I − concentrations, I − enters the thyroid through routes other than NIS. Iodine is an essential constituent of the thyroid hormones (THs), which are phenolic rings joined by an ether link and iodinated at either three positions (3,5,3′-tri-iodo-l-thyronine, or T 3) or four positions (3,5,3′,5′-tetra-i odo-l-thyronine, or T 4). The THs are required for the proper development of the central nervous system 1 , skeleton 2 , and lungs 3 in the fetus and the newborn and for intermediary metabolism at all ages 4. Therefore, both hyper and hypothyroidism are diseased states with serious systemic ramifications. Iodide (I −) is an extremely scarce nutrient in the environment and is supplied only in the diet. The Na + /I − symporter (NIS), a member of solute carrier family 5 (SLC5), is the plasma membrane protein that actively transports I − into the thyroid follicular cells, using as its driving force the Na + gradient generated by the Na + /K + ATPase. NIS transports I − electrogenically with a 2 Na + : 1 I − stoichiometry 1. Since our group cloned the cDNA coding for NIS 5 , we have reported in vivo and in vitro that NIS is regulated transcriptionally and post-transcriptionally by thyroid stimulating hormone (TSH) 6-9. We have identified residues that coordinate NIS substrates 10-12 , and elucidated the basis for the efficient transport of I − by NIS at the physiological (submicromolar) concentrations of I − in the plasma 13. Besides the thyroid gland, NIS is functionally expressed in other tissues including lactating breast 14 , breast cancer 9, 14 , salivary glands 14 , and stomach 1, 9, 14. NIS-mediated I − transport is the key first step in TH biosynthesis, which occurs partly intracellularly and partly in the colloid, an extracellular compartment. I − reaches the colloid through an I − efflux process that has not been fully elucidated. The genetic characterization of patients affected by Pendred syndrome, a disorder characterized by goiter and a partial I − organification defect 15 , led to the identification of pendrin (SLC26A4) as a protein that may mediate I − efflux into the colloid. However, pendrin knockout (KO) mice do not display hypothyroidism 16 , indicating that other mechanisms are also involved in thyroid apical I − efflux. The Na + /monocarboxylate transporter (SMCT) (Slc5a8) is expressed at the apical membrane of thyroid follicular cells 17. SMCT was
The rat thyroid Na ؉ /I ؊ symporter (NIS) was expressed in Xenopus laevis oocytes and characteriz... more The rat thyroid Na ؉ /I ؊ symporter (NIS) was expressed in Xenopus laevis oocytes and characterized using electrophysiological, tracer uptake, and electron microscopic methods. NIS activity was found to be electrogenic and Na ؉-dependent (Na ؉ > > Li ؉ > > H ؉). The apparent affinity constants for Na ؉ and I ؊ were 28 ؎ 3 mM and 33 ؎ 9 M, respectively. Stoichiometry of Na ؉ / anion cotransport was 2:1. NIS was capable of transporting a wide variety of anions (I
BACKGROUND & AIMS: Caudal-related homeodomain transcription factors CDX1 and CDX2 regulate gut de... more BACKGROUND & AIMS: Caudal-related homeodomain transcription factors CDX1 and CDX2 regulate gut development and differentiation of intestinal epithelial cells; they are candidate tumor suppressors of colorectal carcinomas. Because the functions of CDX1 and CDX2 in the colonic epithelium are not fully understood, we sought to identify genes that they target. METHODS: We conducted a chromatin immunoprecipitation (ChIP) screen to identify genes that bind the CDX transcription factors. Expression of target genes was analyzed in colon cells and tissues from Cdx1 Ϫ/Ϫ , Cdx2 ϩ/Ϫ , Apc ϩ/⌬716 , and wild-type (control) mice. RESULTS: Using the ChIP screen, we identified solute carrier family 5, member 8 (SLC5A8, also known as SMCT1) as a direct target of CDX1 and CDX2. CDX transcription factors bind to the promoter region of SLC5A8 and transactivate SLC5A8 reporter constructs. Overexpression of Cdx1 or Cdx2 in human colon cancer cell lines induced expression of endogenous SLC5A8, whereas CDX1 and CDX2 knockdowns reduced its level. Consistently, Slc5a8 expression was significantly reduced in colons of Cdx1 Ϫ/Ϫ or Cdx2 ϩ/Ϫ mice compared with wild-type mice. Slc5a8 levels were also reduced in colonic adenomatous polyps and hamartomas from Apc ϩ/⌬716 and Cdx2 ϩ/Ϫ mutant mice, respectively, compared with adjacent normal colon tissues. CONCLUSIONS: CDX1 and CDX2 bind the promoter region of SLC5A8 and up-regulate its expression in cultured cells and in colonic epithelium. SLC5A8 transports monocarboxylates such as pyruvate, lactate, and butyrate; CDX1 and CDX2 might therefore regulate the uptake of these substances in the colon. C DX genes are human orthologues of Drosophila caudal 1 and code for homeodomain transcription factors. 2 Three CDX family members, CDX1, CDX2, and CDX4, are expressed at early embryonic stages and play important roles in placentation, axis elongation, and hematopoiesis. 3-6 Cdx1 Ϫ/Ϫ mice and Cdx2 ϩ/Ϫ mice suffer homeotic shifts in the skeleton, 3,4,7 whereas Cdx2 Ϫ/Ϫ mouse embryos do not survive beyond 3.5-5.5 days postcoitum, because of abnormal placenta caused by developmental disorders in the trophectoderm.
The rat thyroid Na+/I- symporter (NIS) was expressed in Xenopus laevis oocytes and characterized ... more The rat thyroid Na+/I- symporter (NIS) was expressed in Xenopus laevis oocytes and characterized using electrophysiological, tracer uptake, and electron microscopic methods. NIS activity was found to be electrogenic and Na+-dependent (Na+ > Li+ > H+). The apparent affinity constants for Na+ and I- were 28 +/- 3 mM and 33 +/- 9 microM, respectively. Stoichiometry of Na+/anion cotransport was 2:1. NIS was capable of transporting a wide variety of anions (I-, ClO3-, SCN-, SeCN-, NO3-, Br-, BF4-, IO4-, BrO3-, but perchlorate (ClO4-) was not transported. In the absence of anion substrate, NIS exhibited a Na+-dependent leak current (approximately 35% of maximum substrate-induced current) with an apparent Na+ affinity of 74 +/- 14 mM and a Hill coefficient (n) of 1. In response to step voltage changes, NIS exhibited current transients that relaxed with a time constant of 8-14 ms. Presteady-state charge movements (integral of the current transients) versus voltage relations obey a Bol...
Iodide is an important regulator of thyroid activity. Its excess elicits the Wolff-Chaikoff effec... more Iodide is an important regulator of thyroid activity. Its excess elicits the Wolff-Chaikoff effect, characterized by an acute suppression of thyroid hormone synthesis, which has been ascribed to serum TSH reduction or TGF-β increase and production of iodolipids in the thyroid. These alterations take hours/days to occur, contrasting with the promptness of Wolff-Chaikoff effect. We investigated whether acute iodide administration could trigger events that precede those changes, such as reduction of sodium-iodide symporter (NIS) mRNA abundance and adenylation, and if perchlorate treatment could counteract them. Rats subjected or not to methylmercaptoimidazole treatment (0.03%) received NaI (2,000 μg/0.5 ml saline) or saline intraperitoneally and were killed 30 min up to 24 h later. Another set of animals was treated with iodide and perchlorate, in equimolar doses. NIS mRNA content was evaluated by Northern blotting and real-time PCR, and NIS mRNA poly(A) tail length by rapid amplificat...
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Papers by Nancy Carrasco