Immunolocalization and funtional role of scavenger receptor BI (SR-BI) and ATP-binding cassette A1 (ABC1)

Immunolocalization and funtional role of scavenger receptor BI (SR-BI) and ATP-binding cassette A1 (ABC1)

induction of the colitis. Results are summarized in the table. Furthermore, during the last 2 days of the study, rectal bleeding was significantly hea...

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induction of the colitis. Results are summarized in the table. Furthermore, during the last 2 days of the study, rectal bleeding was significantly heavier in iron supplemented rats and lighter in vitamin E supplemented rats (p-
inflammatory score crypt score Plasma LPO (mmol/ml) Colonic LPO (mmollmg prot) 8-isoprostane (pg/ml) cGPx (mUImg proL) Plasmavitamin C (mmollL)

DSS

iron

vitamin E

Woe+ vitzmin E

2.8 (0.4) 8.2 (0.9) 15.3 (1.33)

4.9 (0.5)a 10.4(09)" 19.3 (1.4)"

2.7 (0.5)b 5.3 (1.1)b 13.8 (1,3)

3.2 (0.4)=" 7 9 (0.9)a, 190 (1.3) a

0 47 (0 06)

2.75 (0.33) '

0.38 (0.1)

2 85 (0.17),

228.3(23.19)

317 5 (41 6) =

243.2(16.2)

224.9(16.1)

135.4(7.3) 53.45(219)

98 4 (7.2)a 45.67(3.00)a

126.1 (14.0) 71 11 (2.12) b

89.7 (2.9)' 54 13(2.22)=b

ABC1 may act as a gatekeeper, and in conditions of hgh cholesterol absorption, it will be upregulated to expulse undesirable sterol In the intestinal lumen. 3667 Dietary Iron Induces Ferroportinl Clustering And Migration into The Basolateral Memlnne Of The Rat Iofeatinal Epithelium Kwo-Yih Yeh, Mary Yeh, Jonathan Glass, LSU Health Science Ctr, Shreveport, LA

a: p_
Background: The molecular mechanisms by which body iron stores regulate intestinal iron absorption are not clear. For absorption across the enterocyte, iron has to across the brush border membrane (BBM), the cell proper and subsequently the basolateral membrane (BLM). Iron transporters located in the BBM and BLM appearto be regulatory targets. Down regulation of the divalent metal transporter in the BBM occurs rapidly after iron exposure (Am J Physio1279:G1070,2000). It is unknown whether the recently identified BLM iron transporter, ferropotinl (FPT1) (Nature 403:776, 2000), also responds to iron. Methods: Rabbit antiserum was generatedagainst a C-terminal 14 amino acid peptide of the rat FPTI. Antiserum specificity was assessed by Western blot analysis of rat FPT1 cDNA ligated to pEGFP-C1 (EGFP-FPT1) and expressed in COS-7 ceils. The anti-FPT1 antiserum reacted only to the fusion protein indicating specificity for FPT1. To determine the effect of iron ingestion on duodenal FPT1 expression, rats were fasted over night, fed 250 mg rodent chow supplemented with iron (50 p.g/g BW), and the expression of FPT1 was subsequently examined. Results: Iron ingestion changed neither the pattern of increased FPT1 expression with cell maturation nor mucosal FPT1 levels. In contrast, ferfitin steadily increased by 3-5 fold at 3 hours after iron feeding. Indirect imrnunoftuorescent staining show that in the unfed (t = O) epithelium FPT1 was mainly in the BLM and formed a "honeycomb" pattern in tangential sections in the area of tight junctions. Weak fluorescence was present m the cytoplasm. With iron feeding FPT1 at the tight junctions decreased in association with the clustering and migration of FPT1 toward the basal region of enterocytes. Conclusions: FPT1 is expressed in the BLM adjacent to the BBM to facilitate in iron transport out of enterocytes. Iron ingestion induced FPT1 clustering and relocation to a region remote from the BBM. FPT1 relocation may either facilitate iron transport out of the cell or be a regulatory mechanism to reduce iron accessibility to FPT1 allowing ferritin to acquire excessiveiron. (Supported by NIH DK-41279 and by the Feist-Weiller Cancer Canter at LSUHSC)

Intestinal Iron Transporter Expression in Liver Disease Katherine A. Stuart, Princess Alexandra Hosp, Brisbane Australia; David M Frazer, David M Purdie, Queensland Institute of Medical Research, Brisbane Australia; Linda M. Fletcher, Princess Atexandra Hnsp, Brisbane Australia; Gregory J. Anderson, Queensland Institute of Medical Research, Brisbane Australia: Darrell H. Crawford, Princess Alexandra Hosp, Brisbane Australia Background: Individuals with end-stage liver disease frequently show an increase in hepatic iron content that is not associated with common mutations in the hemochromatosis gene, HFE. In order to investigate the mechanism of cirrhosis-associated iron overload, we have studied the expression of several recently identified iron transport genes (DMT1, hephaestin, /REG1) in the duodenum of patients with cirrhosis. Methods: Total RNA was extracted from duodenal biopsies of 28 adult patients with cirrhosis (20 patients with hepatocellular diseases and 8 with cholestatk: disease) and 29 control patients with normal liver function tests. The expression of each iron transport gene was assessed by RNaseprotection assays and normalized to the expression of the housekeeping gene GAPDH.Results: Of the cirrhotic patients studied, 24 were classified as iron replete and 4 as iron deficient, while for the control group the numbers were 22 and 7 respectively. No patient was homozygous for the major HFE mutation C282Y. In iron replete subjects, /REGI expression was significantly increased in patients with hepatocellular disease (130.2+60.5, p~0.03), but not those with cholestatic disease (87.4_+20.2, p = 0.81) compared to controls (90.9_+50.5). The expression of DMT1 was not elevated in patients with hepatocellular disease, but was significantly depressed in those with cholestatic disease relative to hepatocelluiar subjects (13.4_+7.8 vs 28.6_+24.0, p = 0.03). In iron deficient cirrhotic patients, both DMT1and/REG1 expression were increased relative to iron replete cirrhotic patients (35.3_+25.4vs 25.4_+22.4, p = 0.54 and 182.0_+92.4 vs 120.9_+56.8, p = 0.08), but the differences were not significant due to the small number of subjects. In iron deficient control subjects, both DMTland/REG1expression were significantly elevated compared to iron replete controls (p=0.gl and 0.009 respectively). Hephaestin expression was not significantly different between groups in any of the comparisons. Conclusions: Alterations in the expression of DMT1and/REG1 in the duodenum of cirrhotic patients may help to explain the elevated iron stores found in patients with advanced liver disease. These iron transport molecules are clearly upregulated in iron deficiency, but how the cirrhotic liver signals the small intestine to affect the expression of these genes remains to be established. The surprisingly low expression of DMTi in patients with cholestatic disease correlates with the lower hepatic iron stores found in this patient group. 3669 The Relative Importance of Luminal and Systemic Signals in the Control of Intestinal Iron Absorption Gregory J. Anderson, Sarah J. Wilkins, Therese M. Murphy, Queensland Institute of Medical Research, Brisbane Australia; Geoffrey J. Cleghorn, Univ of Queensland, Brisbane Australia; David M. Frazer, Queensland Institute of Medical Research, Brisbane Australia Background and Aims: Intestinal iron absorption is regulated predominantly by systemic signals reflecting body iron requirements, but local changes in the iron content of the intestinal lumen and enterocytes can also alter absorption. To investigate the relative contributions of brush border uptake and basolateraltransfer to the control of iron absorption, we have studied how the expression of genes involved in uptake (DMT1) and transfer (hephaestin, IREG1) varies in response to changes in either body iron stores or the intraluminal iron concentration. Methods: Male Sprague-Dawley rats were maintained on iron deficient or iron loaded diets

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