Immunodetection of cyclooxygenase-2 (COX-2) is restricted to tissue macrophages in normal rat liver and to recruited mononuclear phagocytes in liver injury and cholangiocarcinoma

It has been suggested that cyclooxygenase-2 (COX-2)-mediated prostaglandin synthesis is associated with liver inflammation and carcinogenesis. The aim of this study is to identify the cellular source of COX-2 expression in different stages, from acute liver injury through liver fibrosis to cholangiocarcinoma (CC). We induced in rats acute and “chronic” liver injury (thioacetamide (TAA) or carbon tetrachloride (CCl₄)) and CC development (TAA) and assessed COX-2 gene expression in normal and damaged liver tissue by RT-PCR of total RNA. The cellular localization of COX-2 protein in liver tissue was analyzed by immunohistochemistry as well as in isolated rat liver cells by Western blotting. The findings were compared with those obtained in human cirrhotic liver tissue. The specificity of the antibodies was tested by 2-DE Western blot and mass spectrometric identification of the positive protein spots. RT-PCR analysis of total RNA revealed an increase of hepatic COX-2 gene expression in acutely as well as “chronically” damaged liver. COX-2-protein was detected in those ED1⁺/ED2⁺ cells located in the non-damaged tissue (resident tissue macrophages). In addition COX-2 positivity in inflammatory mononuclear phagocytes (ED1⁺/ED2⁻), which were also present within the tumoral tissue was detected. COX-2 protein was clearly detectable in isolated Kupffer cells as well as (at lower level) in isolated “inflammatory” macrophages. Similar results were obtained in human cirrhotic liver. COX-2 protein is constitutively detectable in liver tissue macrophages. Inflammatory mononuclear phagocytes contribute to the increase of COX-2 gene expression in acute and chronic liver damage induced by different toxins and in the CC microenvironment.     

Immunohistochemical studies on the expression pattern of molecular chaperones HSC70 and HSP25 and cell cycle-related proteins cyclin D1 and PCNA in rat liver after thioacetamide intoxication

Intoxication of rats with thioacetamide (TAA) is a model system to investigate mechanisms involved in liver cell death and tissue reconstitution. Our study was undertaken to determine by immunohistochemistry the expression pattern of the cytoprotective chaperone proteins HSC70 and HSP25 and proliferation markers cyclin D1 and PCNA in livers of Wistar rats intraperitoneally injected with TAA at a single dose of 50 mg/kg. For each protein studied we observed distinct dynamic changes in appearance and localization in liver lobules. During 24-36 h after TAA injection the HSC70 cytoplasmic immunoreaction gradually disappeared from hepatocytes localized around central veins and a shift of immunostaining to cell nuclei took place. Then, 36-48 h after TAA injection the HSC70 cytoplasmic immunoreaction reappeared with the highest intensity in hepatocytes surrounding the areas of inflammatory cells. HSP25, undetectable in control hepatocytes began to appear at approximately 36 h after TAA injection and HSP25-immunopositive cells formed a characteristic ring around areas of inflammation. Of the proteins studied, the most rapid reaction to TAA was observed for cyclin D1. As early as 15 min after TAA administration cyclin D1-positive hepatocytes appeared in intermediate and periportal areas of liver lobules and a subsequent shift of staining to centrilobular hepatocytes took place at 36 and 48 h. There was no correlation of cyclin D1 localization either with PCNA-positive cells or mitotic cells. Our observations suggest that in TAA-treated livers HSP25 and HSC70 proteins can play an anti-inflammatory role, and the early and distinct cyclin D1 expression is not related to proliferation of hepatocytes.     

Mesenteric adipose tissue B lymphocytes promote local and hepatic inflammation in non‐alcoholic fatty liver disease mice

Mesenteric adipose tissue (MAT) inflammation is associated with non‐alcoholic fatty liver disease (NAFLD), and immune cells play pivotal roles in the inflammation of adipose tissue. Here, we investigated the roles of MAT B lymphocytes in NAFLD. Mice fed with high‐fat diet (HFD) and normal diet (ND) were killed in time gradients (4, 8 and 12 weeks). Compared with ND‐fed mice, intra‐hepatic CD45⁺CD19⁺ B lymphocytes increased after 4 weeks (P < 0.01) of HFD feeding, and lasted until the 12th week, infiltrated earlier than CD45⁺CD3⁺ T lymphocytes and CD45⁺F4/80⁺ macrophages. The mRNA expression of tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and monocyte chemotactic protein (MCP)‐1 decreased in MAT of B^null HFD‐fed mice compared to that in wild‐type HFD‐fed mice, along with lesser macrophages. Mesenteric adipose tissue B cells from HFD‐fed mice promoted macrophage differentiation to type‐Ι macrophages and expression of pro‐inflammatory cytokines in vitro. Macrophages pre‐treated with MAT B cells from HFD‐fed mice showed elevated mRNA expression of IL‐6 and TNF‐α and declined IL‐10 levels in adipocytes compared to ND MAT B cell pre‐treated macrophages. Besides, internal near‐infrared scanning and external transwell assay showed that HFD MAT B cells migrated to the liver more than ND MAT B cells. High‐fat diet MAT B cells induced higher MCP‐1 and lower IL‐10 expression in primary hepatocytes compared to ND MAT B cells in co‐culture experiment. These data indicate that B lymphocytes infiltrate early in MAT during the development of NAFLD, which may not only promote MAT inflammation by regulating macrophages but also migrate to the liver and induce hepatocytes inflammation.     

HSP70 Enhances Immunosuppressive Function of CD4+CD25+FoxP3+ T Regulatory Cells and Cytotoxicity in CD4+CD25? T Cells

Human CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) control effector T cells and play a central role in peripheral tolerance and immune homeostasis. Heat shock protein 70 (HSP70) is a major immunomodulatory molecule, but its effect on the functions of Tregs is not well understood. To investigate target-dependent and –independent Treg functions, we studied cytokine expression, regulation of proliferation and cytotoxicity after exposure of Tregs to HSP70. HSP70-treated Tregs significantly inhibited proliferation of CD4⁺CD25⁻ target cells and downregulated the secretion of the proinflammatory cytokines IFN-γ and TNF-α. By contrast, HSP70 increased the secretion of Treg suppressor cytokines IL-10 and TGF-β. Treatment with HSP70 enhanced the cytotoxic properties of Tregs only to a minor extent (4-fold), but led to stronger responses in CD4⁺CD25⁻ cells (42-fold). HSP70-induced modulation of T-cell responses was further enhanced by combined treatment with HSP70 plus IL-2. Treatment of Tregs with HSP70 led to phosphorylation of PI3K/AKT and the MAPKs JNK and p38, but not that of ERK1/2. Exposure of Tregs to specific inhibitors of PI3K/AKT and the MAPKs JNK and p38 reduced the immunosuppressive function of HSP70-treated Tregs as indicated by the modified secretion of specific target cell (IFN-γ, TNF-α) and suppressor cytokines (IL-10, TGF-β). Taken together, the data show that HSP70 enhances the suppressive capacity of Tregs to neutralize target immune cells. Thus HSP70-enhanced suppression of Tregs may prevent exaggerated immune responses and may play a major role in maintaining immune homeostasis.     

Synergistic protection by S-adenosylmethionine with vitamins C and E on liver injury induced by thioacetamide in rats

Free radicals are involved in the pathogenesis of acute liver injury induced by thioacetamide (TAA). We investigated the effects of S-adenosylmethionine (SAMe) combined with/without vitamins C and E on TAA-induced acute liver injury in rats. TAA was given intraperitoneally (200 mg kg-1). Antioxidant treatments (SAMe, 25 mg kg-1; vitamin C, 100 mg kg-1; vitamin E, 200 mg kg-1, intraperitoneal) were given 1 h later. Liver histology, enzymology, and ability to release hepatic insulin-sensitizing substance (HISS) were assessed. TAA caused liver tissue injury, increased liver enzymes, and decreased insulin sensitivity (p<0.01). Blockade of HISS release by atropine did not further decrease insulin sensitivity in rats with TAA insult, indicating that the decrease in insulin sensitivity was HISS dependent. Treatment with SAMe alone or vitamins C+E slightly improved liver histology but not the changes in liver enzymes and insulin sensitivity. Combined treatment with SAMe plus vitamins C+E greatly protected the liver from tissue injury, the increase in liver enzymes, and the decrease in insulin sensitivity. In conclusion, acute liver injury causes HISS-dependent insulin resistance (HDIR). There are synergistic antioxidative effects among the antioxidants, SAMe and vitamins C and E, that protect the liver from TAA-induced HDIR, suggesting that antioxidant treatment may best be done using a balanced "cocktail."     

Induction of aldose reductase and polyol dehydrogenase activities in Aureobasidium pullulans by d-xylose,l-arabinose and d-galactose

Summary The induction of aldose reductase and polyol dehydrogenase activities by d-xylose, l-arabinose, d-galactose and d-glucose was studied in the yeast-like organism Aureobasidium pullulans CCY 27-1-26. d-xylose and l-arabinose induced two distinct NADPH-dependent aldose reductases and the inducing saccharide was simultaneously the most efficient substrate for the corresponding enzymatic reaction. Polyol dehydrogenase induced by d-xylose, l-arabinose and d-galactose was strictly NAD⁺-dependent and required only xylitol as a substrate of the enzymatic reaction. l-Arabitol did not act as a substrate for l-arabinose-induced polyol dehydrogenase either in the presence of NAD⁺ or NADP⁺.     

Partial purification of the Na+-dependentd-glucose transport system from renal brush border membranes

Summary A membrane extract enriched with the Na⁺-dependentd-glucose transport system was obtained by differential cholate solubilization of rat renal brush border membranes in the presence of 120mm Na⁺ ions. Sodium ions were essential in stabilizing the transport system during cholate treatment. This membrane extract was further purified with respect to its Na⁺-coupledd-glucose transport activity and protein content by the use of asolectin-equilibrated hydroxylapatite. The reconstituted proteoliposomes prepared from this purified fraction showed a transient accumulation ofd-glucose in response to a Na⁺ gradient. The observed rate of Na⁺-coupledd-glucose uptake by the proteoliposomes represented about a sevenfold increase as compared to that of the reconstituted system derived from an initial 1.2% cholate extract of the membranes. Other Na⁺-coupled transport systems such asl-alanine, -ketoglutarate and phosphate were not detected in these reconstituted proteoliposomes.     

The effect of fluorocitrate on transmitter amino acid release from rat striatal slices

In order to study the role of glutamine from glial cells for the synthesis of transmitter amino acids, the effect of the gliotoxic substance fluorocitrate on amino acid release from slices was investigated. In vivo treatment with 1 nmol fluorocitrate reduced the Ca²⁺ dependent K⁺ evoked release of endogenous glutamate and GABA from the slices, whereas the glutamine efflux decreased and alanine efflux increased. The K⁺ evoked release of [³H]d-aspartate increased during fluorocitrate treatment. The latter is consistent with an inhibited uptake ofd-aspartate into glial cells. Incubation of striatal slices with fluorocitrate (0.1 mM) decreased the glutamine efflux and increased the alanine efflux. Similar to the in vivo condition, fluorocitrate increased the K⁺ evoked [³H]d-asparate release, but the K⁺ evoked release of endogenous glutamate and GABA increased rather than decreased. The ratio between the K⁺ evoked release of exogenousd-aspartate to endogenous glutamate increased in both cases. The results suggest an important role of glial cells in the synthesis and inactivation of transmitter amino acids.Special Issue dedicated to Prof. Holger Hydén.     

Cloning,characterization, and mutational analysis of a highly active and stable <Emphasis Type="SmallCaps">l</Emphasis>-arabinitol 4-dehydrogenase from <Emphasis Type="Italic">Neurospora crassa</Emphasis>

An NAD⁺-dependent l-arabinitol 4-dehydrogenase (LAD, EC 1.1.1.12) from Neurospora crassa was cloned and expressed in Escherichia coli and purified to homogeneity. The enzyme was a homotetramer and contained two Zn²⁺ ions per subunit, displaying similar characteristics to medium-chain sorbitol dehydrogenases (SDHs). High enzymatic activity was observed for substrates l-arabinitol, adonitol, and xylitol and no activity for d-mannitol, d-arabinitol, or d-sorbitol. The enzyme showed strong preference for NAD⁺ but also displayed a very low yet detectable activity with NADP⁺. Mutational analysis of residue F59, the single different substrate-binding residue between LADs and d-SDHs, failed to confer the enzyme the ability to accept d-sorbitol as a substrate, suggesting that the amino acids flanking the active site cleft may be responsible for the different activity and affinity patterns between LADs and SDHs. This enzyme should be useful for in vivo and in vitro production of xylitol and ethanol from l-arabinose. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changes in Extracellular Kynurenic Acid Concentrations in Rat Prefrontal Cortex After <Emphasis Type="SmallCaps">d</Emphasis>-Kynurenine Infusion: An In vivo Microdialysis Study

Using a microdialysis technique, we continuously infused d-kynurenine (KYN) (0, 50, and 100 μM) into the prefrontal cortices (PFCs) of male Sprague–Dawley rats. We then used column-switching high-performance liquid chromatography to assess the alterations in the concentration of kynurenic acid (KYNA)—an antagonist of N-methyl-d-aspartate and α7 nicotinic acetylcholine receptors—in the extracellular fluid in the PFC. Local infusion of d-KYN into the PFC remarkably increased the extracellular KYNA concentration, indicating that d-KYN is metabolized to KYNA in the PFC. The d-KYN-induced increase in KYNA levels was significantly attenuated by the co-administration of 3-methylpyrazole-5-carboxylic acid (AS057278)—a specific inhibitor of d-amino acid oxidase (DAAO). These results suggest that DAAO may be involved in the production of KYNA from d-KYN in the PFC in vivo.     

Plasmid-mediated nicotine degradation inArthrobacter oxidans

The spontaneous loss byArthrobacter oxidans cells of the nicotine-degrading ability (Nic⁺) was 0.06%. It could be increased by treatment with plasmid-curing agents up to 8%. It was possible by conjugation to restore the Nic⁺ phenotype in such cured derivatives and to transfer the Nic⁺ character to Nic^- Arthrobacter species. Plasmid DNA, 160 kb in size as judged by contour length measurements, could be isolated from cleared lysates ofA. oxidans cells by acridine yellow chromatography. Agarose gel electrophoresis of DNA isolated fromArthrobacter exconjugates revealed the occurrence of plasmid DNA within these strains; its mobility was similar to that of the plasmid DNA present inA. oxidans. Although the expression and inducibility of the transferred genes was poor in most of theArthrobacter species exconjugants, apparently authentic 6-hydroxy-l-nicotine oxidase could be identified in these cells after enrichment by an enzyme-specific chromatography.Abbreviations 6-HDNO 6-hydroxy-d-nicotine oxidase - 6-HLNO 6-hydroxy-l-nicotine oxidase - kb kilobase - Nic⁺ ability to usel- ord-nicotine as sole carbon and nitrogen source - Nic^- absence of Nic⁺ character Enzymes (EC 1.5.3.5.) 6-Hydroxy-l-nicotine oxidase, 6-hydroxy-l-nicotine: oxygen oxidoreductase - (EC 1.5.3.6.) 6-hydroxy-d-nicotine oxidase, 6-hydroxy-d-nicotine: oxygen oxidoreductase - (EC 3.1.4.22) ribonuclease A, ribonucleate 3-pyrimidino-oligo-nucleotidohydrolase     

Role of carnosine in preventing thioacetamide-induced liver injury in the rat

Carnosine (beta-alanyl-L-histidine) is a dipeptide with antioxidant properties. Free radicals are involved in the pathogenesis of acute liver injury induced by thioacetamide (TAA). In this study, we investigated the effect of carnosine treatment on TAA-induced oxidative stress and hepatotoxicity. Rats were injected intraperitoneally with TAA (500 mg/kg) and carnosine (250 mg/kg, intraperitoneal) was co-administered with TAA. All animals were killed 24 h after injections. TAA administration resulted in hepatic necrosis, significant increases in plasma transaminase activities as well as hepatic lipid peroxide levels. In addition, hepatic antioxidant system was found to be depressed following TAA administration. When carnosine was co-administered with TAA in rats, plasma transaminase activities were found to approach to normal values in rats. Histological findings also suggested that carnosine has preventive effect on TAA-induced hepatic necrosis. Carnosine treatment caused significant decreases in lipid peroxide levels in TAA-treated rats without any changes in enzymatic and non-enzymatic antioxidants except vitamin E in the liver of rats. Our findings indicate that carnosine, in vivo may have a preventive effect on TAA-induced oxidative stress and hepatotoxicity by acting as an non-enzymatic antioxidant itself.     

The protection by heme oxygenase‐1 induction against thioacetamide‐induced liver toxicity is associated with changes in arginine and asymmetric dimethylarginine

This study was designed to investigate the role of HO‐1 induction in prevention of thioacetamide (TAA)‐induced oxidative stress, inflammation and liver damage. The changes in hepatic dimethylarginine dimethylaminohydrolase (DDAH) activity as well as plasma arginine and asymmetric dimethylarginine (ADMA) levels were also measured to evaluate nitric oxide (NO) bioavailability. Rats were divided into four groups as control, hemin, TAA and hemin + TAA groups. Hemin (50 mg kg^?1, i.p.) was injected to rats 18 h before TAA treatment to induce HO‐1 enzyme expression. Rats were given TAA (300 mg kg^?1, i.p.) and killed 24 h after treatment. Although TAA treatment produced severe hepatic injury, upregulation of HO‐1 ameliorated TAA‐induced liver damage up to some extent as evidence by decreased serum alanine transaminase, aspartate transaminase and arginase activities and histopathological findings. Induction of HO‐1 stimulated antioxidant system and decreased lipid peroxidation in TAA‐treated rats. Myeloperoxidase activity and inducible NO synthase protein expression were decreased, whereas DDAH activity was increased by hemin injection in TAA‐treated rats. Induction of HO‐1 was associated with increased arginine levels and decreased ADMA levels, being the main determinants of NO production, in plasma of TAA‐treated rats. In conclusion, our results indicate that HO‐1 induction alleviated increased oxidative stress and inflammatory reactions together with deterioration in NO production in TAA‐induced liver damage in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Baclofen-induced,calcium-dependent stimulation of in vivo release ofd-[3H]aspartate from rat hippocampus monitored by intracerebral microdialysis

The release ofd-[³H]aspartate (used as a tracer for endogenous glutamate and aspartate) was studied at high K⁺ (100 mM) and under ischemia in rats implanted with 0.3 mm diameter dialysis tubing through the hippocampus. The effect on thed-[³H]aspartate release of the two -aminobutyric acid (GABA) agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) and (±)--(p-chlorophenyl)GABA (baclofen), which specifically activate GABA_A and GABA_B receptors, respectively, was studied. Initial experiments employing HPLC analysis showed a coincident increase in the amounts of glutamate, aspartate and the amount of radioactivity following introduction of K⁺ (100 mM) or a period of ischemia suggesting that thed-[³H]aspartate labels the transmitter pools of the two amino acids under the present experimental conditions. The presence of 10 mM baclofen or 10 mM THIP in the perfusion medium did not inhibit ischemia inducedd-[³H]aspartate release. On the contrary, 10 mM baclofen alone (but not 0.1 or 1 mM) in the perfusion medium induced release ofd-[³H]aspartate in a calcium dependent manner, whereas 10 mM THIP had no significant releasing effect.Special issue dedicated to Dr. Elling Kvamme     

Differential expression of Na<Superscript>+</Superscript>/<Emphasis Type="SmallCaps">d</Emphasis>-glucose cotransport in isolated cells of<Emphasis Type="Italic"> Marsupenaeus japonicus</Emphasis> hepatopancreas

d-Glucose absorptive processes at the gastrointestinal tract of decapod crustaceans are largely under-investigated. We have studied Na⁺-dependent d-glucose transport (Na⁺/d-glucose cotransport) in the hepatopancreas of the Kuruma prawn, Marsupenaeus japonicus, using both brush-border membrane vesicles and purified R and B hepatopancreatic cell suspensions. As assessed by brush-border membrane vesicle studies, Na⁺/d-glucose cotransport was inhibited by phloridzin and responsive to the (inside negative) membrane potential. Furthermore, it was strongly activated by protons (although only in the presence of an inside-negative membrane potential), which correlates with the fact that the lumen of crustacean hepatopancreatic tubules is acidic. When assayed in purified R and B cell suspensions, Na⁺/d-glucose cotransport activity was restricted to B cells only. Mab 13, a monoclonal antibody recognizing an 80- to 85-KDa protein at the brush-border membrane location, inhibited Na⁺/D-glucose cotransport in brush-border membrane vesicles as well as in enriched B cell suspensions. Primers designed after comparison of highly homologous regions of various mammalian sodium-glucose transporter) nucleotide sequences failed to produce RT-PCR amplification products from Kuruma prawn hepatopancreatic RNA. The molecular nature of this Na⁺/d-glucose cotransport system is still to be established.Communicated by: G. Heldmaier     

CXCL10 Controls Inflammatory Pain via Opioid Peptide-Containing Macrophages in Electroacupuncture

Acupuncture is widely used for pain treatment in patients with osteoarthritis or low back pain, but molecular mechanisms remain largely enigmatic. In the early phase of inflammation neutrophilic chemokines direct opioid-containing neutrophils in the inflamed tissue and stimulate opioid peptide release and antinociception. In this study the molecular pathway and neuroimmune connections in complete Freund''s adjuvant (CFA)-induced hind paw inflammation and electroacupuncture for peripheral pain control were analyzed. Free moving Wistar rats with hind paw inflammation were treated twice with electroacupuncture at GB30 (Huan Tiao - gall bladder meridian) (day 0 and 1) and analyzed for mechanical and thermal nociceptive thresholds. The cytokine profiles as well as the expression of opioid peptides were quantified in the inflamed paw. Electroacupuncture elicited long-term antinociception blocked by local injection of anti-opioid peptide antibodies (beta-endorphin, met-enkephalin, dynorphin A). The treatment altered the cytokine profile towards an anti-inflammatory pattern but augmented interferon (IFN)-gamma and the chemokine CXCL10 (IP-10: interferon gamma-inducible protein) protein and mRNA expression with concomitant increased numbers of opioid peptide-containing CXCR3⁺ macrophages. In rats with CFA hind paw inflammation without acupuncture repeated injection of CXCL10 triggered opioid-mediated antinociception and increase opioid-containing macrophages. Conversely, neutralization of CXCL10 time-dependently decreased electroacupuncture-induced antinociception and the number of infiltrating opioid peptide-expressing CXCR3⁺ macrophages. In summary, we describe a novel function of the chemokine CXCL10 - as a regulator for an increase of opioid-containing macrophages and antinociceptive mediator in inflammatory pain and as a key chemokine regulated by electroacupuncture.     

Ontogeny of d-Mannose Transport and Metabolism in Rat Small Intestine

Oral mannose therapy is used to treat congenital disorders of glycosylation caused by a deficiency in phosphomannose isomerase. The segmental distribution and ontogenic regulation of d-mannose transport, phosphomannose isomerase, and phosphomannose mutase is investigated in the small intestine of fetuses, newborn, suckling, 1-month-old, and adult rats. The small intestine transports d-mannose by both Na⁺-dependent and Na⁺-independent transport mechanisms. The activities of both systems normalized to intestinal weight peak at birth and thereafter they decreased. In all the ages tested, the activity of the Na⁺-independent mechanism was higher than that of the Na⁺/mannose transport system. At birth, the Na⁺-independent d-mannose transport in the ileum was significantly higher than that in jejunum. Phosphomannose isomerase activity and mRNA levels increased at 1 month, and the values in the ileum were lower than in jejunum. Phosphomannose mutase activity in jejunum increased during the early stages of life, and it decreased at 1 month old, as does the amount of mannose incorporated into glycoproteins, whereas in the ileum, they were not affected by age. The phosphomannose isomerase/phosphomannose mutase activity ratio decreased at birth and during the suckling period, and increased at 1 month old. In conclusion, intestinal d-mannose transport activity and metabolism were affected by ontogeny and intestinal segment.     

Function and presumed molecular structure of Na+-D-glucose cotransport systems

Functional characterization of Na⁺-d-glucose cotransport in intestine and kidney indicates the existence of heterogeneous Na⁺-d-glucose cotransport systems. Target size analysis of the transporting unit and model analysis of substrate binding have been performed and proteins have been cloned which mediate (SGLT1) and modulate (RS1) the expression of Na⁺-d-glucose cotransport. The experiments support the hypothesis that functional Na⁺-d-glucose cotransport systems in mammals are composed of two SGLT1-type subunits and may contain one or two RS1-type proteins. SGLT1 contains up to twelve membrane-spanning -helices, whereas RS1 is a hydrophilic extracellular protein which is anchored in the brush-border membrane by a hydrophobic -helix at the C-terminus. SGLT1 alone is able to translocate glucose together with sodium; however, RS1 increases the V _max of transport expressed by SGLT1. In addition, the biphasic glucose dependence of transport, which is typical for kidney and has been often observed in intestine, was only obtained after coexpression of SGLT1 and RS1.