Pamela M. Martin

Sodium-coupled Monocarboxylate Transporters in Normal Tissues and in Cancer

SLC5A8 and SLC5A12 are sodium-coupled monocarboxylate transporters (SMCTs), the former being a high-affinity type and the latter a low-affinity type. Both transport a variety of monocarboxylates in a Na+-coupled manner. They are expressed in the gastrointestinal tract, kidney, thyroid, brain, and retina. SLC5A8 is localized to the apical membrane of epithelial cells lining the intestinal tract and proximal tubule. In the brain and retina, its expression is restricted to neurons and the retinal pigment epithelium. The physiologic functions of SLC5A8 include absorption of short-chain fatty acids in the colon and small intestine, reabsorption of lactate and pyruvate in the kidney, and cellular uptake of lactate and ketone bodies in neurons. It also transports the B-complex vitamin nicotinate. SLC5A12 is also localized to the apical membrane of epithelial cells lining the intestinal tract and proximal tubule. In the brain and retina, its expression is restricted to astrocytes and Müller cells. SLC5A8 also functions as a tumor suppressor; its expression is silenced in tumors of colon, thyroid, stomach, kidney, and brain. The tumor-suppressive function is related to its ability to mediate concentrative uptake of butyrate, propionate, and pyruvate, all of which are inhibitors of histone deacetylases. SLC5A8 can also transport a variety of pharmacologically relevant monocarboxylates, including salicylates, benzoate, and γ-hydroxybutyrate. Non-steroidal anti-inflammatory drugs such as ibuprofen, ketoprofen, and fenoprofen, also interact with SLC5A8. These drugs are not transportable substrates for SLC5A8, but instead function as blockers of the transporter. Relatively less is known on the role of SLC5A12 in drug transport.

Butyrate suppresses colonic inflammation through HDAC1-dependent Fas upregulation and Fas-mediated apoptosis of T cells

Butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit protective effects toward inflammatory diseases such as ulcerative colitis (UC) and inflammation-mediated colorectal cancer. Recent studies have shown that chronic IFN-γ signaling plays an essential role in inflammation-mediated colorectal cancer development in vivo, whereas genome-wide association studies have linked human UC risk loci to IFNG, the gene that encodes IFN-γ. However, the molecular mechanisms underlying the butyrate-IFN-γ-colonic inflammation axis are not well defined. Here we showed that colonic mucosa from patients with UC exhibit increased signal transducer and activator of transcription 1 (STAT1) activation, and this STAT1 hyperactivation is correlated with increased T cell infiltration. Butyrate treatment-induced apoptosis of wild-type T cells but not Fas-deficient (Fas(lpr)) or FasL-deficient (Fas(gld)) T cells, revealing a potential role of Fas-mediated apoptosis of T cells as a mechanism of butyrate function. Histone deacetylase 1 (HDAC1) was found to bind to the Fas promoter in T cells, and butyrate inhibits HDAC1 activity to induce Fas promoter hyperacetylation and Fas upregulation in T cells. Knocking down gpr109a or slc5a8, the genes that encode for receptor and transporter of butyrate, respectively, resulted in altered expression of genes related to multiple inflammatory signaling pathways, including inducible nitric oxide synthase (iNOS), in mouse colonic epithelial cells in vivo. Butyrate effectively inhibited IFN-γ-induced STAT1 activation, resulting in inhibition of iNOS upregulation in human colon epithelial and carcinoma cells in vitro. Our data thus suggest that butyrate delivers a double-hit: induction of T cell apoptosis to eliminate the source of inflammation and suppression of IFN-γ-mediated inflammation in colonic epithelial cells, to suppress colonic inflammation.

Blockade of Dendritic Cell Development by Bacterial Fermentation Products Butyrate and Propionate through a Transporter (Slc5a8)-dependent Inhibition of Histone Deacetylases

Mammalian colon harbors trillions of bacteria, yet there is no undue inflammatory response by the host against these bacteria under normal conditions. The bacterial fermentation products acetate, propionate, and butyrate are believed, at least in part, to be responsible for these immunosuppressive effects. Dendritic cells play an essential role in presentation of antigens to T lymphocytes and initiation of adaptive immune responses. Here we report that butyrate and propionate block the generation of dendritic cells from bone marrow stem cells, without affecting the generation of granulocytes. This effect is dependent on the Na+-coupled monocarboxylate transporter Slc5a8, which transports butyrate and propionate into cells, and on the ability of these two bacterial metabolites to inhibit histone deacetylases. Acetate, which is also a substrate for Slc5a8 but not an inhibitor of histone deacetylases, does not affect dendritic cell development, indicating the essential role of histone deacetylase inhibition in the process. The blockade of dendritic cell development by butyrate and propionate is associated with decreased expression of the transcription factors PU.1 and RelB. Butyrate also elicits its biologic effects through its ability to activate the G-protein-coupled receptor Gpr109a, but this mechanism is not involved in butyrate-induced blockade of dendritic cell development. The participation of Slc5a8 and the non-involvement of Gpr109a in butyrate effects have been substantiated using bone marrow cells obtained from Slc5a8−/− and Gpr109a−/− mice. These findings uncover an important mechanism underlying the anti-inflammatory functions of the bacterial fermentation products butyrate and propionate.

In vivo protection against retinal neurodegeneration by sigma receptor 1 ligand (+)-pentazocine.

PURPOSE To evaluate the neuroprotective properties of the sigma receptor 1 (sigmaR1) ligand, (+)-pentazocine in an in vivo model of retinal neurodegeneration. METHODS Spontaneously diabetic Ins2(Akita/+) and wild-type mice received intraperitoneal injections of (+)-pentazocine for 22 weeks beginning at diabetes onset. Retinal mRNA and protein were analyzed by RT-PCR and Western blot analysis. Retinal histologic sections were measured to determine total retinal thickness, thicknesses of inner-outer nuclear and plexiform layers (INL, ONL, IPL, INL), and the number of cell bodies in the ganglion cell layer (GCL). Immunolabeling experiments were performed using antibodies specific for 4-hydroxynonenal and nitrotyrosine, markers of lipid peroxidation, and reactive nitrogen species, respectively, and an antibody specific for vimentin to view radial Müller fibers. RESULTS sigmaR1 mRNA and protein levels in the Ins2(Akita/+) retina were comparable to those in the wild-type, indicating that sigmaR1 is an available target during the disease process. Histologic evaluation of eyes of Ins2(Akita/+) mice showed disruption of retinal architecture. By 17 to 25 weeks after birth, Ins2(Akita/+) mice demonstrated approximately 30% and 25% decreases in IPL and INL thicknesses, respectively, and a 30% reduction in ganglion cells. In the (+)-pentazocine-treated group, retinas of Ins2(Akita/+) mice showed remarkable preservation of retinal architecture; IPL and INL thicknesses of (+)-pentazocine-treated Ins2(Akita/+) mouse retinas were within normal limits. The number of ganglion cells was 15.6 +/- 1.5 versus 10.4 +/- 1.2 cells/100 mum retinal length in (+)-pentazocine-treated versus nontreated mutant mice. Levels of nitrotyrosine and 4-hydroxynonenal increased in Ins2(Akita/+) retinas, but were reduced in (+)-pentazocine-treated mice. Retinas of Ins2(Akita/+) mice showed loss of the uniform organization of radial Müller fibers. Retinas of (+)-pentazocine-treated mice maintained the radial organization of glial processes. CONCLUSION Sustained (+)-pentazocine treatment in an in vivo model of retinal degeneration conferred significant neuroprotection, reduced evidence of oxidative stress, and preserved retinal architecture, suggesting that sigmaR1 ligands are promising therapeutic agents for intervention in neurodegenerative diseases of the retina.

Identity of SMCT1 (SLC5A8) as a neuron-specific Na+-coupled transporter for active uptake of L-lactate and ketone bodies in the brain.

SMCT1 is a sodium‐coupled (Na+‐coupled) transporter for l‐lactate and short‐chain fatty acids. Here, we show that the ketone bodies, β‐d‐hydroxybutyrate and acetoacetate, and the branched‐chain ketoacid, α‐ketoisocaproate, are also substrates for the transporter. The transport of these compounds via human SMCT1 is Na+‐coupled and electrogenic. The Michaelis constant is 1.4 ± 0.1 mm for β‐d‐hydroxybutyrate, 0.21 ± 0.04 mm for acetoacetate and 0.21 ± 0.03 mm for α‐ketoisocaproate. The Na+ : substrate stoichiometry is 2 : 1. As l‐lactate and ketone bodies constitute primary energy substrates for neurons, we investigated the expression pattern of this transporter in the brain. In situ hybridization studies demonstrate widespread expression of SMCT1 mRNA in mouse brain. Immunofluorescence analysis shows that SMCT1 protein is expressed exclusively in neurons. SMCT1 protein co‐localizes with MCT2, a neuron‐specific Na+‐independent monocarboxylate transporter. In contrast, there was no overlap of signals for SMCT1 and MCT1, the latter being expressed only in non‐neuronal cells. We also demonstrate the neuron‐specific expression of SMCT1 in mixed cultures of rat cortical neurons and astrocytes. This represents the first report of an Na+‐coupled transport system for a major group of energy substrates in neurons. These findings suggest that SMCT1 may play a critical role in the entry of l‐lactate and ketone bodies into neurons by a process driven by an electrochemical Na+ gradient and hence, contribute to the maintenance of the energy status and function of neurons.

Transporters and receptors for short-chain fatty acids as the molecular link between colonic bacteria and the host.

The mutually beneficial relationship between colonic bacteria and the host has been recognized but the molecular aspects of the relationship remain poorly understood. Dietary fiber is critical to this relationship. The short-chain fatty acids acetate, propionate and butyrate, generated by bacterial fermentation of dietary fiber, serve as messengers between colonic bacteria and the host. The beneficial effects of these bacterial metabolites in colon include, but are not limited to, suppression of inflammation and prevention of cancer. Recent studies have identified the plasma membrane transporter SLC5A8 and the cell-surface receptors GPR109A and GPR43 as essential for the biologic effects of short-chain fatty acids in colon. These three proteins coded by the host genome provide the molecular link between colonic bacteria and the host.

Transport of Nicotinate and Structurally Related Compounds by Human SMCT1 (SLC5A8) and Its Relevance to Drug Transport in the Mammalian Intestinal Tract

PurposeTo examine the involvement of human SMCT1, a Na+-coupled transporter for short-chain fatty acids, in the transport of nicotinate/structural analogs and monocarboxylate drugs, and to analyze its expression in mouse intestinal tract.Materials and MethodsWe expressed human SMCT1 in X. laevis oocytes and monitored its function by [14C]nicotinate uptake and substrate-induced inward currents. SMCT1 expression in mouse intestinal tract was examined by immunofluorescence.Results[14C]Nicotinate uptake was several-fold higher in SMCT1-expressing oocytes than in water-injected oocytes. The uptake was inhibited by short-chain/medium-chain fatty acids and various structural analogs of nicotinate. Exposure of SMCT1-expressing oocytes to nicotinate induced Na+-dependent inward currents. Measurements of nicotinate flux and associated charge transfer into oocytes suggest a Na+:nicotinate stoichiometry of 2:1. Monocarboxylate drugs benzoate, salicylate, and 5-aminosalicylate are also transported by human SMCT1. The transporter is expressed in the small intestine as well as colon, and the expression is restricted to the lumen-facing apical membrane of intestinal and colonic epithelial cells.ConclusionsHuman SMCT1 transports not only nicotinate and its structural analogs but also various monocarboxylate drugs. The transporter is expressed on the luminal membrane of the epithelial cells lining the intestinal tract. SMCT1 may participate in the intestinal absorption of monocarboxylate drugs.

SIRT1 is essential for oncogenic signaling by estrogen/estrogen receptor α in breast cancer

The NAD-dependent histone deacetylase silent information regulator 1 (SIRT1) is overexpressed and catalytically activated in a number of human cancers, but recent studies have actually suggested that it may function as a tumor suppressor and metastasis inhibitor in vivo. In breast cancer, SIRT1 stabilization has been suggested to contribute to the oncogenic potential of the estrogen receptor α (ERα), but SIRT1 activity has also been associated with ERα deacetylation and inactivation. In this study, we show that SIRT1 is critical for estrogen to promote breast cancer. ERα physically interacted and functionally cooperated with SIRT1 in breast cancer cells. ERα also bound to the promoter for SIRT1 and increased its transcription. SIRT1 expression induced by ERα was sufficient to activate antioxidant and prosurvival genes in breast cancer cells, such as catalase and glutathione peroxidase, and to inactivate tumor suppressor genes such as cyclin G2 (CCNG2) and p53. Moreover, SIRT1 inactivation eliminated estrogen/ERα-induced cell growth and tumor development, triggering apoptosis. Taken together, these results indicated that SIRT1 is required for estrogen-induced breast cancer growth. Our findings imply that the combination of SIRT1 inhibitors and antiestrogen compounds may offer more effective treatment strategies for breast cancer.

Cloning and functional identification of slc5a12 as a sodium-coupled low-affinity transporter for monocarboxylates (SMCT2).

We report in the present paper, on the isolation and functional characterization of slc5a12, the twelfth member of the SLC5 gene family, from mouse kidney. The slc5a12 cDNA codes for a protein of 619 amino acids. Heterologous expression of slc5a12 cDNA in mammalian cells induces Na+-dependent transport of lactate and nicotinate. Several other short-chain monocarboxylates compete with nicotinate for the cDNA-induced transport process. Expression of slc5a12 in Xenopus oocytes induces electrogenic and Na+-dependent transport of lactate, nicotinate, propionate and butyrate. The substrate specificity of slc5a12 is similar to that of slc5a8, an Na+-coupled transporter for monocarboxylates. However, the substrate affinities of slc5a12 were much lower than those of slc5a8. slc5a12 mRNA is expressed in kidney, small intestine and skeletal muscle. In situ hybridization with sagittal sections of mouse kidney showed predominant expression of slc5a12 in the outer cortex. This is in contrast with slc5a8, which is expressed in the cortex as well as in the medulla. The physiological function of slc5a12 in the kidney is likely to mediate the reabsorption of lactate. In the intestinal tract, slc5a12 is expressed in the proximal parts, whereas slc5a8 is expressed in the distal parts. The expression of slc5a12 in the proximal parts of the intestinal tract, where there is minimal bacterial colonization, suggests that the physiological function of slc5a12 is not to mediate the absorption of short-chain monocarboxylates derived from bacterial fermentation but rather to mediate the absorption of diet-derived short-chain monocarboxylates. Based on the functional and structural similarities between slc5a8 and slc5a12, we suggest that the two transporters be designated as SMCT1 (sodium-coupled monocarboxylate transporter 1) and SMCT2 respectively.

Hepcidin expression in mouse retina and its regulation via lipopolysaccharide/Toll-like receptor-4 pathway independent of Hfe

Hepcidin is a hormone central to the regulation of iron homeostasis in the body. It is believed to be produced exclusively by the liver. Ferroportin, an iron exporter, is the receptor for hepcidin. This transporter/receptor is expressed in Müller cells, photoreceptor cells and the RPE (retinal pigment epithelium) within the retina. Since the retina is protected by the retinal-blood barriers, we asked whether ferroportin in the retina is regulated by hepcidin in the circulation or whether the retina produces hepcidin for regulation of its own iron homeostasis. Here we show that hepcidin is expressed robustly in Müller cells, photoreceptor cells and RPE cells, closely resembling the expression pattern of ferroportin. We also show that bacterial LPS (lipopolysaccharide) is a regulator of hepcidin expression in Müller cells and the RPE, both in vitro and in vivo, and that the regulation occurs at the transcriptional level. The action of LPS on hepcidin expression is mediated by the TLR4 (Toll-like receptor-4). The upregulation of hepcidin by LPS occurs independent of Hfe (human leukocyte antigen-like protein involved in Fe homeostasis). The increase in hepcidin levels in retinal cells in response to LPS treatment is associated with a decrease in ferroportin levels. The LPS-induced upregulation of hepcidin and consequent down-regulation of ferroportin is associated with increased oxidative stress and apoptosis within the retina in vivo. We conclude that retinal iron homeostasis may be regulated in an autonomous manner by hepcidin generated within the retina and that chronic bacterial infection/inflammation of the retina may disrupt iron homeostasis and retinal function.