Expression profiling and immunolocalization of Na+-D-glucose-cotransporter 1 in mice employing knockout mice as specificity control indicate novel locations and differences between mice and rats.

The expression and localization of sodium-D-glucose cotransporter SGLT1 (SLC5A1), which is involved in small intestinal glucose absorption and renal glucose reabsorption, is of high biomedical relevance because SGLT1 inhibitors are currently tested for antidiabetic therapy. In human and rat organs, detailed expression profiling of SGLT1/Sglt1 mRNA and immunolocalization of the transporter protein has been performed. Using polyspecific antibodies and preabsorption with antigenic peptide as specificity control, in several organs, different immunolocalizations of SGLT1/Sglt1 between human and rat were obtained. Because the preabsorption control does not exclude cross-reactivity with similar epitopes, some localizations remained ambiguous. In the present study, we performed an immunocytochemical localization of Sglt1 in various organs of mice. Specificities of the immunoreactions were evaluated using antibody preabsorption with the Sglt1 peptide and the respective organs of Sglt1 knockout mice. Because staining in some locations was abolished after antibody preabsorption but remained in the knockout mice, missing staining in knockout mice was used as specificity criterion. The immunolocalization in mouse was identical or similar to rat in many organs, including small intestine, liver, and kidney. However, the male-dominant renal Sglt1 protein expression in mice differed from the female-dominant expression in rats, and localization in lung, heart, and brain observed in rats was not detected in mice. In mice, several novel locations of Sglt1, e.g., in eyes, tongue epithelial cells, pancreatic ducts, prostate, and periurethral glands were detected. Using end-point and quantitative RT-PCR in various organs, different Sglt1 expression in mice and rats was confirmed.

SGLT1 Deficiency Turns Listeria Infection into a Lethal Disease in Mice.

BACKGROUND: Cellular glucose uptake may involve either non-concentrative glucose carriers of the GLUT family or Na+-coupled glucose-carrier SGLT1, which accumulates glucose against glucose gradients and may thus accomplish cellular glucose uptake even at dramatically decreased extracellular glucose concentrations. SGLT1 is not only expressed in epithelia but as well in tumour cells and immune cells. Immune cell functions strongly depend on their metabolism, therefore we hypothesized that deficiency of SGLT1 modulates the defence against bacterial infection. To test this hypothesis, we infected wild type mice and gene targeted mice lacking functional SGLT1 with Listeria monocytogenes. METHODS: SGLT1 deficient mice and wild type littermates were infected with 1x104 CFU Listeria monocytogenes intravenously. Bacterial titers were determined by colony forming assay, SGLT1, TNF-α, IL-6 and IL-12a transcript levels were determined by qRT-PCR, as well as SGLT1 protein abundance and localization by immunohistochemistry. RESULTS: Genetic knockout of SGLT1 (Slc5a1-/- mice) significantly compromised bacterial clearance following Listeria monocytogenes infection with significantly enhanced bacterial load in liver, spleen, kidney and lung, and significantly augmented hepatic expression of TNF-α and IL-12a. While all wild type mice survived, all SGLT1 deficient mice died from the infection. CONCLUSIONS: SGLT1 is required for bacterial clearance and host survival following murine Listeria infection.

Long-term effects of melatonin and resveratrol on aging rats: A multi-biomarker approach.

Aging-related impaired body structure and functions may be, at least partially, caused by elevated oxidative stress. Melatonin (MEL) and resveratrol (RSV) may act as antioxidant and anti-aging compounds, but these actions in experimental animals and humans are controversial. Herein, a rat model of aging was used to study the long-term sex-related effects of MEL and RSV treatment on body mass and blood/plasma parameters of DNA damage, oxidative status (glutathione and malondialdehyde levels), and concentrations of sex hormones. Starting from the age of 3mo, for the next 9mo or 21mo male and female Wistar rats (n = 4-7 per group) were given water to drink (controls) or 0.1 % ethanol in water (vehicle), or MEL or RSV (each 10 mg/L vehicle). DNA damage in whole blood cells was tested by comet assay, whereas in plasma, glutathione, malondialdehyde, and sex hormones were determined by established methods. Using statistical analysis of data by ANOVA/Scheffe post hoc, we observed a similar sex- and aging-dependent rise of body mass in both sexes and drop of plasma testosterone in control and vehicle-treated male rats, whose pattern remained unaffected by MEL and RSV treatment. Compared with controls, all other parameters remained largely unchanged in aging and differently treated male and female rats. We concluded that the sex- and aging-related pattern of growth and various blood parameters in rats were not affected by the long-term treatment with MEL and RSV at the estimated daily doses (300-400 µg/kg b.m.) that exceed usual moderate consumption in humans.

CD13 is a critical regulator of cell-cell fusion in osteoclastogenesis.

The transmembrane aminopeptidase CD13 is highly expressed in cells of the myeloid lineage, regulates dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. Here, we show that CD13-deficient mice present a low bone density phenotype with increased numbers of osteoclasts per bone surface, but display a normal distribution of osteoclast progenitor populations in the bone marrow and periphery. In addition, the bone formation and mineral apposition rates are similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Lack of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells containing remarkably high numbers of nuclei. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP1 must be downregulated for fusion to proceed, these are aberrantly sustained at high levels even in CD13-deficient mature multi-nucleated osteoclasts. Further, the stability of fusion-promoting proteins is maintained in the absence of CD13, implicating CD13 in protein turnover mechanisms. Together, we conclude that CD13 may regulate cell-cell fusion by controlling the expression and localization of key fusion regulatory proteins that are critical for osteoclast fusion.

Sex-dependent expression of metallothioneins MT1 and MT2 and concentrations of trace elements in rat liver and kidney tissues: Effect of gonadectomy.

Metallothioneins (MTs) exhibit binding affinity for several essential and toxic trace elements. Previous studies in rodents indicated sex differences in the hepatic and renal expression of MTs and concentrations of various elements. The mechanism responsible for these differences has not been resolved. Here, in the liver and kidney tissues of sham-operated and gonadectomized male and female rats we determined the expression of MT1 and MT2 (MT1&2) mRNA by RT-PCR, abundance of MT1&2 proteins by Western blotting and immunocytochemistry, concentrations of essential (Fe, Zn, Cu, Co) and toxic (Cd, Hg, Pb) elements by ICP-MS, and oxidative status parameters (SOD, GPx, MDA, GSH) by biochemical methods. In both organs, the expression of MT1&2 mRNA and MT1&2 proteins was female-dominant, upregulated by castration, and downregulated by ovariectomy. Concentrations of Fe in the liver and Co in the kidneys followed the same pattern. Most other elements (Zn, Cu, Cd, Hg) exhibited female- or male-dominant sex differences, affected by gonadectomy in one or both organs. Pb was sex- and gonadectomy-unaffected. GPx and MDA were elevated and associated with the highest concentrations of Fe only in the female liver. We conclude that the sex-dependent expression of MT1&2 mRNA and proteins in the rat liver and kidneys may include different mechanisms. In the liver, the female-dominant tissue concentrations of Fe may generate oxidative stress which is a potent enhancer of MTs production, whereas in kidneys, the female-dominant expression of MTs may be unrelated to Fe-mediated oxidative stress.

Sodium-glucose cotransporters: new targets of cancer therapy?

Glucose, the key source of metabolic energy, is imported into cells by two categories of transporters: 1) facilitative glucose transporters (GLUTs) and 2) secondary active sodium-glucose cotransporters (SGLTs). Cancer cells have an increased demand for glucose uptake and utilisation compared to normal cells. Previous studies have demonstrated the overexpression of GLUTs, mainly GLUT1, in many cancer types. As the current standard positron emission tomography (PET) tracer 2-deoxy-2-(18F)fluoro-D-glucose (2-FDG) for imaging tumour cells via GLUT1 lacks in sensitivity and specificity, it may soon be replaced by the newly designed, highly sensitive and specific SGLT tracer α-methyl-4-(F-18)fluoro-4-deoxy-Dglucopyranoside (Me-4FDG) in clinical detection and tumour staging. This tracer has recently demonstrated the functional activity of SGLT in pancreatic, prostate, and brain cancers. The mRNA and protein expression of SGLTs have also been reported in colon/colorectal, lung, ovarian, head, neck, and oral squamous carcinomas. So far, SGLTs have been poorly investigated in cancer, and their protein expression and localisation are often controversial due to a lack of specific SGLT antibodies. In this review, we describe current knowledge concerning SGLT1 and SGLT2 (over)expression in various cancer types. The findings of SGLTs in malignant cells may help in developing novel cancer therapies with SGLT2 or SGLT1/SGLT2 inhibitors already used in diabetes mellitus treatment.

Apigenin: A dietary flavonoid with diverse anticancer properties.

Apigenin is a natural flavonoid found in several dietary plant foods such as vegetables and fruits. A large number of studies conducted over the past years have shown that this particular natural compound has potential antioxidant, anti-inflammatory, and anticancer properties. Therefore, apigenin has generated a great deal of interest as a possible chemotherapeutic modality due to its low intrinsic toxicity and remarkable effects on normal versus cancerous cells, compared with other structurally related flavonoids. Here, we review its role in anticancer research, as well as several cancer signalling pathways, including MAPK, PI3K/Akt and NF-κB pathways, and their specific role in different cancer types. Based on the available literature, the beneficial effects of apigenin as a future anticancer modality are promising but they require further in vitro and in vivo studies to enable its translation from bench to bedside.