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Ctr1 (copper transporter 1) mediates high-affinity copper uptake. Ctr2 (copper transporter 2) shares sequence similarity with Ctr1, yet its function in mammalian cells is poorly understood. In African green monkey kidney COS-7 cells and rat tissues, Ctr2 migrated as a predominant band of approximately 70 kDa and was most abundantly expressed in placenta and heart. A transiently expressed hCtr2-GFP (human Ctr2-green fluorescent protein) fusion protein and the endogenous Ctr2 in COS-7 cells were mainly localized to the outer membrane of cytoplasmic vesicles, but were also detected at the plasma membrane. Biotinylation of Ctr2 with the membrane-impermeant reagent sulfo-NHS-SS-biotin [sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate] confirmed localization at the cell surface. Cells expressing hCtr2-GFP hyperaccumulated copper when incubated in medium supplemented with 10 microM CuSO(4), whereas cells depleted of endogenous Ctr2 by siRNAs (small interfering RNAs) accumulated lower levels of copper. hCtr2-GFP expression did not affect copper efflux, suggesting that hCtr2-GFP increased cellular copper concentrations by promoting uptake at the cell surface. Kinetic analyses showed that hCtr2-GFP stimulated saturable copper uptake with a K(m) of 11.0+/-2.5 microM and a K(0.5) of 6.9+/-0.7 microM when data were fitted to a rectangular hyperbola or Hill equation respectively. Competition experiments revealed that silver completely inhibited hCtr2-GFP-dependent copper uptake, whereas zinc, iron and manganese had no effect on uptake. Furthermore, increased copper concentrations in hCtr2-GFP-expressing cells were inversely correlated with copper chaperone for Cu/Zn superoxide dismutase protein expression. Collectively, these results suggest that Ctr2 promotes copper uptake at the plasma membrane and plays a role in regulating copper levels in COS-7 cells.

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The dominant faecal flora of the rat was determined using randomly cloned 16S rDNA comparative sequence analysis. A total of 109 near full-length 16S rDNA clones were sequenced, representing 69 unique 16S rRNA phylotypes or operational taxonomic units (OTUs). Estimates of species richness indicated that approximately 338 species were present in the faeces, suggesting that only 20% of species were identified. Only two of 39 Gram-negative clones aligned with previously cultured species, the remainder fell into a separate lineage within the Bacteroides-Cytophaga phylum. Several clones within this new group were related to 16S rDNA sequences previously identified from mouse faeces. Lactobacilli were the most abundant Gram-positive species, representing 23% of the total clones but only 7% of OTUs. The remaining Gram-positive clones were distributed among the Clostridium coccoides group (9%), the Clostridium leptum subgroup (18%), and throughout the low GC Gram-positive bacteria (13%). The majority of OTUs (63/69 or 91%) were less than 97% homologous to previously cultured bacteria. Faecal samples were also cultured using a variety of anaerobic media. With the exception of the lactobacilli, the cultured isolates demonstrated low species diversity and poorly reflected the population, as defined through comparative sequence analysis.

We are exposed to millions of microbial and dietary antigens via the gastrointestinal tract, which likely play a key role in type 1 diabetes (T1D). We differentiated the effects of these two major environmental factors on gut immunity and T1D. Diabetes-prone BioBreeding (BBdp) rats were housed in specific pathogen-free (SPF) or germ-free (GF) conditions and weaned onto diabetes-promoting cereal diets or a protective low-antigen hydrolyzed casein (HC) diet, and T1D incidence was monitored. Fecal microbiota 16S rRNA genes, immune cell distribution, and gene expression in the jejunum were analyzed. T1D was highest in cereal-SPF (65%) and cereal-GF rats (53%) but inhibited and delayed in HC-fed counterparts. Nearly all HC-GF rats remained diabetes-free, whereas HC-fed SPF rats were less protected (7 vs. 29%). Bacterial communities differed in SPF rats fed cereal compared with HC. Cereal-SPF rats displayed increased gut CD3+ and CD8α+ lymphocytes, ratio of Ifng to Il4 mRNA, and Lck expression, indicating T-cell activation. The ratio of CD3+ T cells expressing the Treg marker Foxp3+ was highest in HC-GF and lowest in cereal-SPF rats. Resident CD163+ M2 macrophages were increased in HC-protected rats. The cathelicidin antimicrobial peptide (Camp) gene was upregulated in the jejunum of HC diet–protected rats, and CAMP+ cells colocalized with CD163. A cereal diet was a stronger promoter of T1D than gut microbes in association with impaired gut immune homeostasis.

BackgroundMechanistic data to support health claims is often generated using rodent models, and the influence of prebiotic supplementation has largely been evaluated using male rodents. Given that sex-based differences in immune parameters are well recognized and recent evidence suggests differences in microbiota composition between sexes, validation of the effectiveness of prebiotics merits assessment in both males and females. Here, we have compared the effect of oligofructose (OF) supplementation on the fecal bacterial community, short chain fatty acid profiles, and gut mucosal and systemic immune parameters in male and female rats.MethodsMale and female rats were fed rodent chow or chow supplemented with OF (5 % w/w). Fecal community change was examined by analyzing 16S rRNA gene content. To compare effects of OF between sexes at the gut microbial and mucosal immune level, fecal short chain fatty acid and tissue cytokine profiles were measured. Serum lipopolysaccharide levels were also evaluated by the limulus amebocyte lysate assay as an indirect means of determining gut permeability between sexes.ResultsIn the fecal community of females, OF supplementation altered community structure by increasing abundance in the Phylum Bacteroidetes. In male rats, no changes in fecal community structure were observed, although fecal butyrate levels significantly increased. Liver Immunoglobulin A (IgA) levels were higher in males relative to females fed OF, and serum LPS concentrations were higher in males independent of diet. Females had higher basal levels of the regulatory cytokine interleukin-10 (IL-10) in the colon and liver, while males had higher basal levels of the pro-inflammatory cytokines IL-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1) in the cecum and liver.ConclusionsWe have shown that male and female rat gut communities metabolize an OF-supplemented diet differently. Sex-specific responses in both the fecal community and systemic immune parameters suggest that this difference may result from an increase in the availability of gut peptidyl-nitrogen in the males. These findings demonstrate the importance of performing sex-comparative studies when investigating potential health effects of prebiotics using rodent models.

The shifts in 25(OH)D distribution brought about by standardization indicate its importance in drawing correct conclusions about potential population deficiencies and insufficiencies and in permitting the comparison of distributions between national surveys.

Djungarian hamsters ( Phodopus sungorus) acclimated to a short photoperiod (8:16-h light-dark cycle) display spontaneous daily torpor with ad libitum food availability. The time course of body temperature (Tb), metabolic rate, respiratory quotient (RQ), and substrate and enzyme changes was measured during entrance into torpor and in deep torpor. RQ, blood glucose, and serum lipids are high during the first hours of torpor but then gradually decline, suggesting that glucose is the primary fuel during the first hours of torpor, with a gradual change to lipid utilization. No major changes in enzyme activities were observed during torpor except for inactivation of the pyruvate dehydrogenase (PDH) complex in liver, brown adipose tissue, and heart muscle. PDH inactivation closely correlates with the reduction of total metabolic rate, whereas in brain, kidney, diaphragm, and skeletal muscle, PDH activity was maintained at the initial level. These findings suggest inhibition of carbohydrate oxidation in heart, brown adipose tissue, and liver during entrance into daily torpor.