Journal of Nutrition Biochemical, Molecular, and Genetic Mechanisms

Anemic Copper-Deficient Rats, but Not Mice, Display Low Hepcidin Expression and High Ferroportin Levels [Biochemical, Molecular, and Genetic Mechanisms]

Jenkitkasemwong, S., Broderius, M., Nam, H., Prohaska, J. R., Knutson, M. D. — Fri, 19 Mar 2010 09:01:14 PDT

The transmembrane protein ferroportin (Fpn) is essential for iron efflux from the liver, spleen, and duodenum. Fpn is regulated predominantly by the circulating iron regulatory hormone hepcidin, which binds to cell surface Fpn, initiating its degradation. Accordingly, when hepcidin concentrations decrease, Fpn levels increase. A previous study found that Fpn levels were not elevated in copper-deficient (CuD) mice that had anemia, a condition normally associated with dramatic reductions in hepcidin. Lack of change in Fpn levels may be because CuD mice do not display reduced concentrations of plasma iron (holotransferrin), a modulator of hepcidin expression. Here, we examined Fpn protein levels and hepcidin expression in CuD rats, which exhibit reduced plasma iron concentrations along with anemia. We also examined hepcidin expression in anemic CuD mice with normal plasma iron levels. We found that CuD rats had higher liver and spleen Fpn levels and markedly lower hepatic hepcidin mRNA expression than did copper-adequate (CuA) rats. In contrast, hepcidin levels did not differ between CuD and CuA mice. To examine potential mediators of the reduced hepcidin expression in CuD rats, we measured levels of hepatic transferrin receptor 2 (TfR2), a putative iron sensor that links holotransferrin to hepcidin production, and transcript abundance of bone morphogenic protein 6 (BMP6), a key endogenous positive regulator of hepcidin production. Diminished hepcidin expression in CuD rats was associated with lower levels of TfR2, but not BMP6. Our data suggest that holotransferrin and TfR2, rather than anemia or BMP6, are signals for hepcidin synthesis during copper deficiency.

Cholecalciferol Supplementation throughout Winter Does Not Affect Markers of Bone Turnover in Healthy Young and Elderly Adults [Biochemical, Molecular, and Genetic Mechanisms]

Fri, 19 Feb 2010 09:01:12 PST

Although there have been several studies of the effect of vitamin D status on bone turnover in the elderly, the findings are unclear, and, furthermore, to date very few have investigated this in young adults. The objective of these randomized, placebo-controlled, double-blind, 2-center intervention studies was to investigate the effect of cholecalciferol supplementation (0, 5, 10, and 15 µg cholecalciferol/d) throughout wintertime on indices of vitamin D status and bone turnover in young (aged 20–40 y; n = 215) and elderly (aged ≥64 y; n = 204) adults, with relatively high mean calcium intakes of 976 and 874 mg/d, respectively. Fasting serum concentrations of 25-hydroxyvitamin D [25(OH)D], parathyroid hormone (PTH), osteocalcin, bone-specific alkaline phosphatase, and carboxyterminal collagen crosslinks were measured by enzyme immunoassays at baseline and endpoint. Fok I and Taq I vitamin D receptor (VDR) genotypes were determined by real-time PCR. Endpoint serum 25(OH)D increased (P < 0.0001) in a dose-related manner with increasing supplemental cholecalciferol (up to 15 µg/d) in 20–40-y olds and up to 10 µg/d in ≥64-y olds. Endpoint serum PTH was lower (P < 0.05) in the 3 cholecalciferol-supplemented groups compared with that in the placebo group in ≥64-y olds, but cholecalciferol supplementation did not affect other markers in either cohort and there was no significant interaction with VDR genotype. In conclusion, cholecalciferol supplementation alone throughout winter did not affect bone turnover markers in apparently healthy young and elderly adults, even when stratified by VDR genotype.

Early Weaning Reduces Small Intestinal Alkaline Phosphatase Expression in Pigs [Biochemical, Molecular, and Genetic Mechanisms]

Lackeyram, D., Yang, C., Archbold, T., Swanson, K. C., Fan, M. Z. — Fri, 19 Feb 2010 09:01:12 PST

Expression of the small intestinal alkaline phosphatase (IAP) is enterocyte differentiation dependent and plays essential roles in the detoxification of pathogenic bacterial lipopolysaccharide endotoxin, maintenance of luminal pH, organic phosphate digestion, and fat absorption. This study was conducted to examine the effect of early weaning on adaptive changes in IAP digestive capacity (V_cap) and IAP gene expression compared with suckling counterparts in pigs at ages 10–22 d. Weaning decreased (P < 0.05) IAP enzyme affinity by 26% and IAP maximal enzyme activity by 22%, primarily in the jejunal region, with the jejunum expressing 84–86% of the whole gut mucosal IAP V_cap [mol/(kg body weight·d)]. The majority (98%) of the jejunal mucosal IAP maximal activity was associated with the apical membrane and the remaining (2%) existed as the intracellular soluble IAP. Weaning reduced the abundance of the 60-kDa IAP protein associated with the proximal jejunal apical membrane by 64% (P < 0.05). Furthermore, weaning reduced (P < 0.05) the relative abundance of the proximal jejunal IAP mRNA by 58% and this was in association with decreases (P < 0.05) in the abundances of cytoplasmic (27%) and nuclear (29%) origins of IAP caudal-associated homeobox transcription factor 1. In conclusion, early weaning decreased small intestinal IAP V_cap, IAP catalytic affinity, and IAP gene expression, and this may in part contribute to the susceptibility of early-weaned piglets to increased occurrence of enteric diseases and growth-check.

Hepatic Acyl-Coenzyme A:Cholesterol Acyltransferase-2 Expression Is Decreased in Mice with Hyperhomocysteinemia [Biochemical, Molecular, and Genetic Mechanisms]

Devlin, A. M., Singh, R., Bottiglieri, T., Innis, S. M., Green, T. J. — Wed, 20 Jan 2010 09:03:14 PST

Alterations in lipid metabolism may contribute to the pathology of hyperhomocysteinemia (HHcy). Our objective in this study was to test the hypothesis that HHcy is associated with changes in liver acyl CoA:cholesterol acyl transferase 2 (ACAT2) expression and cholesteryl esters (CE) in mice with HHcy. ACAT2 is encoded by Soat2 and functions to catalyze the esterification of cholesterol with acyl-CoA. Mice heterozygous for disruption of the cystathionine-β-synthase gene (Cbs +/–) and C57BL/6 mice (Cbs +/+) were fed a control diet or a diet high in l-methionine (8.60 g/kg) and low in folic acid (0.20 mg/kg) to induce HHcy (HH diet). Lower Soat2 mRNA (P < 0.05) and ACAT protein (P < 0.001), higher total oleic acid [18:1(n-9)], and lower CE 18:1(n-9) was found in liver from Cbs +/– mice fed the HH diet, with higher plasma total homocysteine concentrations, than Cbs +/+ mice fed the control diet (35.01 ± 5.6 vs. 2.21 ± 0.6 µmol/L, respectively). In silico searches identified a CpG-rich region in the 5' portion of the Soat2 gene, which was differentially methylated (P < 0.05) in Cbs +/– mice fed the HH diet than in Cbs +/+ mice fed the control diet and was accompanied by higher (P < 0.05) B1 repeat element methylation, an indicator of global de novo methylation. These findings show altered methylation and expression of Soat2/ACAT2 in liver from mice with HHcy and suggest a role for changes in liver CE in the pathology of HHcy.

Leptin Receptor Polymorphisms Interact with Polyunsaturated Fatty Acids to Augment Risk of Insulin Resistance and Metabolic Syndrome in Adults [Biochemical, Molecular, and Genetic Mechanisms]

Wed, 20 Jan 2010 09:03:14 PST

The leptin receptor (LEPR) is associated with insulin resistance, a key feature of metabolic syndrome (MetS). Gene-fatty acid interactions may affect MetS risk. The objective was to investigate the relationship among LEPR polymorphisms, insulin resistance, and MetS risk and whether plasma fatty acids, a biomarker of dietary fatty acids, modulate this. LEPR polymorphisms (rs10493380, rs1137100, rs1137101, rs12067936, rs1805096, rs2025805, rs3790419, rs3790433, rs6673324, and rs8179183), biochemical measurements, and plasma fatty acid profiles were determined in the LIPGENE-SU.VI.MAX study of MetS cases and matched controls (n = 1754). LEPR rs3790433 GG homozygotes had increased MetS risk compared with the minor A allele carriers [odds ratio (OR) = 1.65; 95% CI: 1.05–2.57; P = 0.028], which may be accounted for by their increased risk of elevated insulin concentrations (OR 2.40; 95% CI: 1.28–4.50; P = 0.006) and insulin resistance (OR = 2.15; 95% CI: 1.18–3.90; P = 0.012). Low (less than median) plasma (n-3) and high (n-6) PUFA status exacerbated the genetic risk conferred by GG homozygosity to hyperinsulinemia (OR 2.92–2.94) and insulin resistance (OR 3.40–3.47). Interestingly, these associations were abolished against a high (n-3) or low (n-6) PUFA background. Importantly, we replicated some of these findings in an independent cohort. Homozygosity for the LEPR rs3790433 G allele was associated with insulin resistance, which may predispose to increased MetS risk. Novel gene-nutrient interactions between LEPR rs3790433 and PUFA suggest that these genetic influences were more evident in individuals with low plasma (n-3) or high plasma (n-6) PUFA.

Antiangiogenic Effects of Indole-3-Carbinol and 3,3'-Diindolylmethane Are Associated with Their Differential Regulation of ERK1/2 and Akt in Tube-Forming HUVEC [Biochemical, Molecular, and Genetic Mechanisms]

Kunimasa, K., Kobayashi, T., Kaji, K., Ohta, T. — Fri, 18 Dec 2009 09:01:33 PST

We previously reported that indole-3-carbinol (I3C), found in cruciferous vegetables, suppresses angiogenesis in vivo and in vitro. However, the underlying molecular mechanisms still remain unclear. Antiangiogenic effects of its major metabolite, 3,3'-diindolylmethane (DIM), also have not been fully elucidated. In this study, we investigated the effects of these indoles on angiogenesis and tested a hypothesis that I3C and DIM inhibit angiogenesis and induce apoptosis by affecting angiogenic signal transduction in human umbilical vein endothelial cells (HUVEC). We found that I3C and DIM at 25 µmol/L significantly inhibited tube formation and only DIM induced a significant increase in apoptosis in tube-forming HUVEC. DIM showed a stronger antiangiogenic activity than I3C. At the molecular level, I3C and DIM markedly inactivated extracellular signal-regulated kinase 1/2 (ERK1/2) and the inhibitory effect of DIM was significantly greater than that of I3C. DIM treatment also resulted in activation of the caspase pathway and inactivation of Akt, whereas I3C did not affect them. These results indicate that I3C and DIM had a differential potential in the regulation of the 2 principal survival signals, ERK1/2 and Akt, in endothelial cells. We also demonstrated that pharmacological inhibition of ERK1/2 and/or Akt was enough to inhibit tube formation and induce caspase-dependent apoptosis in tube-forming HUVEC. We conclude that both I3C and DIM inhibit angiogenesis at least in part via inactivation of ERK1/2 and that inactivation of Akt by DIM is responsible for its stronger antiangiogenic effects than those of I3C.

A Bifidobacterium Probiotic Strain and Its Soluble Factors Alleviate Chloride Secretion by Human Intestinal Epithelial Cells [Biochemical, Molecular, and Genetic Mechanisms]

Heuvelin, E., Lebreton, C., Bichara, M., Cerf-Bensussan, N., Heyman, M. — Fri, 18 Dec 2009 09:01:33 PST

Previous studies indicate that certain probiotic bacterial strains or their soluble products can alleviate proinflammatory cytokine secretion by intestinal epithelial cells (IEC), but their impact on epithelial chloride (Cl^–) secretion remains elusive. To further decipher the mechanisms of the cross-talk between bacteria/soluble factors and epithelial cells, we analyzed the capacity of the probiotic strain Bifidobacterium breve C50 (Bb C50), its conditioned medium, and other commensal Gram (+) bacteria to modulate epithelial Cl^– secretion. The effect of Bb C50 on carbachol- (CCh) or forskolin (Fsk)-induced Cl^– secretion was measured in an IEC line in Ussing chambers. The mechanisms involved in the regulation of Cl^– secretion were assessed by measuring intracellular Ca²⁺ concentration, phosphatase activity, protein kinase (PK) C and PKA activation, and cystic fibrosis transmembrane conductance regulator (CFTR) expression. CCh- or Fsk-induced Cl^– secretion [short-circuit current (Isc): 151 ± 28 and 98 ± 14 µA/cm², respectively] was inhibited dose-dependently by Bb C50 (Isc 33 ± 12 and 49 ± 7 µA/cm² at multiplicity of infection 100; P < 0.02). Fsk-induced Cl^– secretion was also inhibited by Lactobacillus rhamnosus 10893. No other inhibitory effect was recorded with the other Gram (+) bacteria tested. The inhibitory effect of Bb C50 on CCh-induced Cl^– secretion targeted a step downstream of epithelial Ca²⁺ mobilization and was associated with decreased PKC activity. Thus, Bb C50 and secreted soluble factors, by inhibiting phosphorylation processes, may promote intestinal homeostasis by controlling Cl^– secretion.