Diabetes was induced in male Lewis rats by a single injection of streptozocin (50 mg/kg body wt ip). After 10-14 days, diabetic and age- and sex-matched control animals were killed, and their proximal small intestines were removed. Villus-tip, mid-villus, and lower-villus enterocytes were harvested from each group with a method that combined divalent cation chelation with mild mechanical dissociation. These fractions were used as starting material to prepare brush-border membrane vesicles. Preparations from each of these fractions were then analyzed and compared with respect to their Na(+)-gradient-dependent and Na(+)-independent D-glucose transport, lipid fluidity, and lipid composition. The results of these experiments demonstrated that 1) maximum rates of Na(+)-gradient-dependent D-glucose transport (Vmax) were greatest in membrane vesicles prepared from mature cells (villus tip and mid villus) of control rats; 2) the glucose concentration producing half-maximal rates of transport (Km), however, was significantly lower in lower-villus membrane vesicles of control rats, suggesting that a distinct glucose transporter existed in the membranes of these relatively immature enterocytes; 3) Na(+)-gradient-dependent, but not Na(+)-independent, D-glucose uptake was greater in diabetic membrane vesicles prepared from mid-villus and lower-villus fractions but not in vesicles prepared from villus-tip cells; and 4) no obvious relationship between alterations in membrane lipid fluidity and enhanced uptake of Na(+)-gradient-dependent D-glucose by these transporter(s) could be established in this experimental model of acute diabetes mellitus.
The current studies were undertaken to establish an in vitro cellular model to study the transport of SO[Formula: see text] and Cl − and hormonal regulation and to define the possible function of the downregulated in adenoma ( DRA) gene. Utilizing a postconfluent Caco-2 cell line, we studied the OH − gradient-driven 35 SO[Formula: see text] and 36 Cl − uptake. Our findings consistent with the presence of an apical carrier-mediated 35 SO[Formula: see text]/OH − exchange process in Caco-2 cells include: 1) demonstration of saturation kinetics [Michaelis-Menten constant ( K m ) of 0.2 ± 0.08 mM for SO[Formula: see text] and maximum velocity of 1.1 ± 0.2 pmol · mg protein −1 · 2 min −1 ]; 2) sensitivity to inhibition by DIDS ( K i = 0.9 ± 0.3 μM); and 3) competitive inhibition by oxalate and Cl − but not by nitrate and short chain fatty acids, with a higher K i (5.95 ± 1 mM) for Cl − compared with oxalate ( K i = 0.2 ± 0.03 mM). Our results also suggested that the SO[Formula: see text]/OH − and Cl − /OH − exchange processes in Caco-2 cells are distinct based on the following: 1) the SO[Formula: see text]/OH − exchange was highly sensitive to inhibition by DIDS compared with Cl − /OH − exchange activity ( K i for DIDS of 0.3 ± 0.1 mM); 2) Cl − competitively inhibited the SO[Formula: see text]/OH − exchange activity with a high K i compared with the K m for SO[Formula: see text], indicating a lower affinity for Cl − ; 3) DIDS competitively inhibited the Cl − /OH − exchange process, whereas it inhibited the SO[Formula: see text]/OH − exchange activity in a mixed-type manner; and 4) utilizing the RNase protection assay, our results showed that 24-h incubation with 100 nM of thyroxine significantly decreased the relative abundance of DRA mRNA along with the SO[Formula: see text]/OH − exchange activity but without any change in Cl − /OH − exchange process. In summary, these studies demonstrated the feasibility of utilizing Caco-2 cell line as a model to study the apical SO[Formula: see text]/OH − and Cl − /OH − exchange processes in the human intestine and indicated that the two transporters are distinct and that DRA may be predominantly a SO[Formula: see text]transporter with a capacity to transport Cl − as well.
S terol R esponse E lement B inding P rotein 2 (SREBP2) transcription factor is a master regulator of cholesterol homeostasis. Treatment with statins, inhibitors of cholesterol synthesis, activates intestinal SREBP2 that may hinder their cholesterol‐lowering effects. Overactivation of SREBP2 in mouse liver was shown to have no effect on plasma cholesterol. However, the influence of activating intestinal SREBP2 on plasma cholesterol is not known. We have generated a novel transgenic mouse model with intestine specific overexpression of active SREBP2 (ISR2) driven by villin promoter. ISR2 mice showed overexpression of active SREBP2 specifically in the intestine. Microarray analysis of jejunal RNA from ISR2 mice showed a significant increase in genes involved in fatty acid and cholesterol synthesis. Cholesterol (Chol) and triglyceride (TG) in jejunum and liver (mg/g protein) were significantly increased in ISR2 vs wild type mice (wt) (Chol: 20.2±1.5 vs 15.9±1.2; TG: 82.3±14.9 vs 30.4±4.9; Chol: 40.5±3.1 vs 22.7±0.9; TG: 83.1±3.1 vs. 50.8±3.6, respectively). Serum Chol was significantly increased in VLDL and LDL fractions in ISR2 vs wt mice (15.92±2.3 vs 5.8±2 and 50.36±5 vs 16.05±0.5 mg/dl, respectively). In conclusion, activation of intestinal SREBP2 alone seems to be sufficient to increase plasma cholesterol, highlighting the essential role of intestine in maintaining cholesterol homeostasis in the body. (Supported by VA and NIDDK).
Abstract Serotonin transporter (SERT) plays a critical role in regulating extracellular availability of serotonin (5-HT) in the gut and brain. Mice with deletion of SERT develop metabolic syndrome as they age. Changes in the gut microbiota are being increasingly implicated in Metabolic Syndrome and Diabetes. To investigate the relationship between the gut microbiome and SERT, this study assessed the fecal and cecal microbiome profile of 11 to 12 week-old SERT +/+ and SERT −/− mice. Microbial DNA was isolated, processed for metagenomics shotgun sequencing, and taxonomic and functional profiles were assessed. 34 differentially abundant bacterial species were identified between SERT +/+ and SERT −/− . SERT −/− mice displayed higher abundances of Bacilli species including genera Lactobacillus, Streptococcus, Enterococcus , and Listeria . Furthermore, SERT −/− mice exhibited significantly lower abundances of Bifidobacterium species and Akkermansia muciniphilia . Bacterial community structure was altered in SERT −/− mice. Differential abundance of bacteria was correlated with changes in host gene expression. Bifidobacterium and Bacilli species exhibited significant associations with host genes involved in lipid metabolism pathways. Our results show that SERT deletion is associated with dysbiosis similar to that observed in obesity. This study contributes to the understanding as to how changes in gut microbiota are associated with metabolic phenotype seen in SERT deficiency.
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