R. S. Fry

Ctr1 Is an Apical Copper Transporter in Mammalian Intestinal Epithelial Cells in Vivo That Is Controlled at the Level of Protein Stability

Copper is an essential trace element that functions in a diverse array of biochemical processes that include mitochondrial respiration, neurotransmitter biogenesis, connective tissue maturation, and reactive oxygen chemistry. The Ctr1 protein is a high-affinity Cu+ importer that is structurally and functionally conserved in yeast, plants, fruit flies, and humans and that, in all of these organisms, is localized to the plasma membrane and intracellular vesicles. Although intestinal epithelial cell-specific deletion of Ctr1 in mice demonstrated a critical role for Ctr1 in dietary copper absorption, some controversy exists over the localization of Ctr1 in intestinal epithelial cells in vivo. In this work, we assess the localization of Ctr1 in intestinal epithelial cells through two independent mechanisms. Using immunohistochemistry, we demonstrate that Ctr1 localizes to the apical membrane in intestinal epithelial cells of the mouse, rat, and pig. Moreover, biotinylation of intestinal luminal proteins from mice fed a control or a copper-deficient diet showed elevated levels of both total and apical membrane Ctr1 protein in response to transient dietary copper limitation. Experiments in cultured HEK293T cells demonstrated that alterations in the levels of the glycosylated form of Ctr1 in response to copper availability were a time-dependent, copper-specific posttranslational response. Taken together, these results demonstrate apical localization of Ctr1 in intestinal epithelia across three mammalian species and suggest that increased Ctr1 apical localization in response to dietary copper limitation may represent an adaptive response to homeostatically modulate Ctr1 availability at the site of intestinal copper absorption.

High dietary iron reduces transporters involved in iron and manganese metabolism and increases intestinal permeability in calves

A 56-d experiment was designed to examine the effect of high dietary Fe on metal transporters involved in Fe and Mn metabolism. Fourteen weaned Holstein calves were stratified by weight and randomly assigned to 1 of 2 treatments: 1) no supplemental Fe (normal Fe) or 2) 750mg of supplemental Fe/kg of dry matter (high Fe). Jugular blood was collected on d 0, 35, and 56. At the end of the trial, 6 calves per treatment were humanely killed and duodenal scrapings, liver, and heart were collected for analysis. Additionally, proximal duodenum was mounted on Ussing chambers to assess intestinal barrier integrity. Calves receiving high dietary Fe displayed decreased transepithelial resistance and increased apical-to-basolateral flux of radiolabeled mannitol, suggesting that high Fe created increased intestinal permeability. Feeding calves a diet high in Fe decreased average daily gain, dry matter intake, and feed efficiency. Hemoglobin and serum Fe concentrations did not differ due to dietary treatment. High dietary Fe increased concentrations of Fe in the liver, but did not affect heart or duodenal Fe concentrations. Duodenal Mn concentrations were lowered by feeding a high Fe diet, but liver and heart Mn concentrations were not affected. As determined by real-time reverse transcription PCR, relative hepatic expression of the gene that encodes the Fe regulatory hormone hepcidin was 5-fold greater in calves fed high dietary Fe. Hepcidin is released in response to increased Fe status and binds to the Fe export protein ferroportin causing ferroportin to be degraded, thereby reducing dietary Fe absorption. Confirmation of this result was achieved through Western blotting of duodenal protein, which revealed that ferroportin was decreased in calves fed high dietary Fe. Duodenal protein expression of divalent metal transporter 1 (DMT1), a Fe import protein that can also transport Mn, tended to be reduced by high dietary Fe. Transcript levels of several genes involved in Fe metabolism in liver and duodenum were unchanged by treatment. In summary, feeding calves a diet high in Fe induced a signal cascade (hepcidin) designed to reduce absorption of Fe (via reduced protein expression of ferroportin and DMT1) in a manner similar to that reported in rodents. Additionally, reduced levels of DMT1 protein appeared to decrease duodenal Mn, suggesting that Mn may also be a substrate for DMT1 in cattle.

Chromium propionate enhances insulin sensitivity in growing cattle

Thirty-six Angus and Angus×Simmental heifers, averaging 291 kg, were used to determine the effects of dietary Cr, in the form of Cr propionate (Cr Prop), on glucose metabolism and serum insulin concentrations following glucose administration. Heifers were stratified by body weight (BW) within a breed and randomly assigned to treatments. Treatments consisted of 0, 3, 6, or 9 mg of supplemental Cr/d from Cr Prop. Based on dry matter (DM) intakes, the daily doses of Cr were equivalent to 0.47, 0.94, and 1.42 mg of supplemental Cr/kg of DM. Heifers were individually fed a corn silage-based diet at a level of 2% of BW. Each heifer was also fed 0.45 kg of a ground corn supplement daily that served as a carrier for supplemental Cr. Glucose tolerance tests were performed on d 44 of the study. Glucose was infused via jugular catheters at a level of 0.45 g/kg of BW(0.75) over a course of 1 to 2 min. Blood samples were collected at -10, 0, 5, 10, 15, 30, 45, 60, 90, 120, 150, and 180 min relative to glucose dosing for glucose and insulin determination. Area under the glucose response curve was lower (1,603 vs. 1,964 mg/dL per minute) in heifers supplemented with Cr from 0 to 45 min following glucose challenge. Serum insulin concentrations were lower in Cr-supplemented heifers than in controls following glucose infusion. The molar ratio of insulin to glucose was also lower in Cr-supplemented heifers relative to controls. Serum insulin and serum insulin to glucose ratios did not differ among heifers supplemented with 3, 6, or 9 mg of Cr/d. Results indicate that Cr Prop supplementation increased tissue sensitivity to insulin in growing heifers. Based on insulin sensitivity, Cr requirements (as Cr Prop) of growing heifers can be met by supplementing with 3 mg of Cr/d or 0.47 mg of Cr/kg of DM.

Tolerance of cattle to increased dietary sulfur and effect of dietary cation-anion balance

The objective of this study was to determine if dietary cation-anion balance (DCAB) affects the concentration of S that can be tolerated by growing and finishing cattle without adversely affecting performance. Angus cross and Bradford steers (n=114; average initial BW=252.8 kg) were blocked by BW and breed, and randomly assigned within a block to treatment. The design was a 3 × 2 factorial arrangement of treatments with S (from NH(4)SO(4)) supplemented at 0, 0.15, or 0.30% of DM, and NaHCO(3) added at 0 or 1.0% of DM. Each treatment consisted of 3 pens containing 5 steers and 1 pen containing 4 steers. Steers were used in an 84-d growing study followed by a finishing study. A corn silage-based diet was fed during the growing study and a corn-based diet was fed during the finishing study. Steers were not randomized between experiments. The analyzed concentrations of S in the growing diets were 0.12, 0.30, and 0.46%, whereas the analyzed concentrations of S in the finishing diets were 0.13, 0.31, and 0.46% for treatments supplemented with 0, 0.15, and 0.30% S, respectively. Increasing DCAB by approximately 15 mEq/100 g of DM, by the addition of NaHCO(3,) did not affect (P > 0.36) performance during the growing or finishing studies. During the growing study DMI was not affected (P=0.29) by dietary S. Steers fed diets containing 0.30% S had greater ADG (P=0.02) and G:F (P=0.01) than those receiving 0.46% S, but similar (P > 0.36) performance to steers fed 0.12% S. During the finishing study, steers fed diets containing 0.46% S had less ADG than steers fed 0.13 (P=0.004) or 0.31% S (P=0.07), whereas ADG did not differ (P=0.18) among steers fed 0.13 and 0.31% S. Steers fed diets containing 0.31 (P=0.01) or 0.46% S (P=0.001) had less DMI than controls, but G:F was not affected (P=0.52) by S during the finishing study. Carcass characteristics did not differ (P > 0.18) among steers fed diets containing 0.13 and 0.31% S. Steers receiving diets containing 0.46% S had decreased HCW (P=0.001), quality (P=0.02), and yield grades (P=0.04) than steers receiving 0.13% S. Plasma Cu concentrations on d 101 of the finishing phase and liver Cu concentrations at slaughter were greater (P ≤ 0.05) in control steers compared with those fed diets containing 0.31 or 0.46% S. This study indicates that steers fed growing diets can tolerate up to 0.46% S with minimum effects on performance. Finishing steers tolerated diets containing 0.31% S without adverse affects on ADG or G:F. However, 0.46% S greatly decreased ADG and DMI, and increasing DCAB did not prevent these depressions.

The addition of high manganese to a copper-deficient diet further depresses copper status and growth of cattle

A study was conducted evaluating the effect of long-term Cu deficiency, with or without high Mn, on growth, gene expression and Cu status of beef cattle. Twenty-one Angus calves were born to cows receiving one of the following treatments: (1) 10 mg supplemental Cu/kg DM (+Cu); (2) no supplemental Cu and 2 mg Mo/kg DM ( - Cu); (3) - Cu diet plus 500 mg supplemental Mn/kg DM ( - Cu+Mn). Calves were weaned at approximately 183 d of age and individually fed throughout the growing and finishing phases. Plasma Cu was lower (P < 0.01) in - Cu calves compared with +Cu calves while high dietary Mn further depressed (P < 0.01) plasma Cu in - Cu+Mn calves v. - Cu calves. Liver Cu concentrations in +Cu calves were greater (P < 0.01) than in - Cu calves, with no differences between - Cu and - Cu+Mn calves. The daily body-weight gain of +Cu calves was greater (P < 0.01) than - Cu calves during the period from birth to weaning, but did not differ during the growing phase. - Cu+Mn calves gained less (P < 0.05) than - Cu calves during the growing phase. DM intake was lower (P < 0.01) in - Cu+Mn calves v. - Cu calves, and did not differ among +Cu and - Cu calves. The relative gene expression of cytochrome c oxidase in the liver was lower (P < 0.05) in - Cu calves compared with +Cu or - Cu+Mn calves. In conclusion, feeding a Cu - deficient diet in combination with high Mn negatively affected the growth and Cu status of beef cattle.

Amount and source of dietary copper affects small intestine morphology, duodenal lipid peroxidation, hepatic oxidative stress,and mRNA expression of hepatic copper regulatory proteins in weanling pigs 1 2

Thirty weanling, crossbred barrows (SUS SCROFA) were used to determine the effects of amount and source of dietary Cu on small intestinal morphology and lipid peroxidation, Cu metabolism, and mRNA expression of proteins involved in hepatic Cu homeostasis. At 21 d of age, pigs were stratified by BW (6.33 ± 0.23 kg) and allocated to 1 of the following dietary treatments: i) control (no supplemental Cu; 6.7 mg Cu/kg), ii) 225 mg supplemental Cu/kg diet from Cu sulfate (CuSO(4)), or iii) 225 mg supplemental Cu/kg diet from tribasic Cu chloride (TBCC). Pigs were housed 2 pigs per pen and were fed a 3-phase diet regimen until d 35 or 36 of the study. During harvest, bile and liver were obtained for mineral analysis, and liver samples were also obtained for analysis of liver glutathione (GSH) and mRNA expression of Cu regulatory proteins. Segments of duodenum, proximal jejunum, and ileum were obtained for mucosal morphology, and duodenal mucosal scrapings were collected from all pigs for analysis of malondialdehyde (MDA). Duodenal villus height was reduced in CuSO(4) pigs compared with control (P = 0.001) and TBCC (P = 0.03) pigs. Villus height in the proximal jejunum of CuSO(4) pigs was reduced (P = 0.03) compared with control pigs, but ileal villus height was not affected (P = 0.82) by treatment. Duodenal MDA concentrations were greater (P = 0.03) in CuSO(4) pigs and tended to be greater (P = 0.10) in pigs supplemented with TBCC compared with control pigs. Liver Cu was greater (P = 0.01) in CuSO(4) vs. control pigs, and tended (P = 0.07) to be greater in TBCC pigs than control pigs. Bile Cu concentrations were greater (P < 0.001) in CuSO(4) and TBCC pigs vs. controls and were also greater (P = 0.04) in TBCC vs. CuSO(4) pigs. Total liver GSH concentrations were less (P = 0.02) in pigs fed diets supplemented with CuSO(4) vs. pigs fed control diets but total liver GSH did not differ (P = 0.11) between control and TBCC pigs. Hepatic mRNA of cytochrome c oxidase assembly protein 17 was less (P = 0.01) in CuSO(4) and tended to be less (P = 0.08) in TBCC pigs vs. control pigs. Expression of antioxidant 1 mRNA was greater (P = 0.04) in TBCC pigs and tended to be greater (P = 0.06) in CuSO(4) pigs compared with control pigs. Results of this study indicated that, when fed at 225 mg Cu/kg diet, TBCC may cause less oxidative stress in the duodenum than CuSO(4). Feeding weanling pigs increased Cu resulted in modulation of certain Cu transporters and chaperones at the transcription level.

Effects of supplementing dairy cows with chromium propionate on milk and tissue chromium concentrations.

Eight primiparous and 8 multiparous Holstein cows were used to determine the effects of Cr supplementation, in the form of Cr propionate (Cr Prop), on milk and tissue Cr concentrations. Cows were randomly assigned by parity to one of 2 diets: 1) control diet or 2) 2 mg of supplemental Cr/kg of DM. The level of Cr Prop supplemented exceeded by 4-fold the concentration of 0.5 mg of Cr/kg permitted by the FDA. Experimental diets were fed from approximately 30 d prepartum until at least 91 d postpartum, resulting in a minimum of 121 d of exposure to supplemental Cr. The control prepartum and postpartum diets analyzed 0.48 and 0.38 mg of Cr/kg of DM, respectively. Milk samples were obtained from the a.m. milking on d 0 (colostrum), 7, 14, 21, 28, 42, 56, 77, and 90 and on the final day of the study for Cr analysis. Cows were harvested after lactating for a minimum of 91 d and samples of liver, kidney, semitendinosus muscle, and fat were obtained for Cr analysis. Chromium was measured using electrothermal atomic absorption spectrophotometry. Milk Cr concentration averaged 1.7 ng/mL and was affected by day of lactation but not by Cr or a Cr × day interaction. Supplementation of 2 mg of Cr/kg of DM increased kidney Cr by approximately 3-fold and liver Cr concentrations by approximately 2-fold. Chromium concentrations in muscle and fat were not affected by Cr supplementation. In summary, supplementation of Cr Prop at a level of 2 mg of Cr/kg of DM did not affect Cr concentration in milk, muscle, or fat, the major bovine products consumed by humans.

Effect of dietary copper and breed on gene products involved in copper acquisition, distribution, and use in Angus and Simmental cows and fetuses

Copper (Cu) deficiency is a widespread problem in cattle across the United States and breed differences in Cu metabolism may contribute to this issue. Intracellular Cu is tightly regulated by transport and chaperone proteins, and to date, these mechanisms have not been elucidated to address breed differences in Cu metabolism, nor have these proteins been characterized in bovine fetal liver. Mature, pregnant Angus (n = 8) and Simmental (n = 8) cows (∼4 mo into gestation) were used in a 2 × 2 factorial arrangement of treatments. All cows were bred to Angus sires resulting in an Angus vs. Simmental × Angus comparison for fetuses. Cows were randomly assigned to corn silage-based diets that were either adequate (+Cu) or deficient (-Cu; 6.6 mg Cu/kg DM) in Cu. Diets were individually fed for 112 d. At the end of the study, cows were harvested to collect duodenal mucosa scrapes, liver samples, and fetal liver samples for mineral analysis and also for mRNA and protein analysis of Cu transport and chaperone proteins. Placentomes were also obtained for mineral analysis. Plasma Cu and liver Cu were affected by Cu, breed, and Cu × breed. Both of these Cu indices were less (P ≤ 0.05) in-Cu Simmentals (-CuS) than in-Cu Angus (- uA), but were similar among +Cu Simmental (+CuS) and +Cu Angus cows (+CuA). Duodenal Cu was less (P = 0.01) in-Cu vs. +Cu cows. Placentome Cu was less (P = 0.003) in-Cu vs. +Cu cows, and was also less (P = 0.03) in Simmentals vs. Angus. Fetal liver Cu was less (P = 0.002) in-Cu vs. +Cu fetuses, and was also less (P = 0.05) in Simmental × Angus vs. Angus. Abundance of Cu transporter1 (CTR1) protein and transcripts for Cu transporters and chaperones were not affected by Cu or breed in liver and were not affected by Cu in the intestine. Duodenal Ctr1 was less (P = 0.04) and CTR1 tended (P = 0.10) to be less in Simmentals vs. Angus. Expression of Atp7a tended (P = 0.08) to be less in Simmentals than in Angus. In fetal liver, expression of antioxidant 1 (Atox1), cytochrome c oxidase assembly protein 17 (Cox17), and Cu metabolism MURR1 domain 1 (Commd1) were up-regulated (P ≤ 0.05) in-Cu vs. +Cu fetuses. In conclusion, less expression of duodenal Ctr1 and a tendency for less CTR1 (P = 0.10) and Atp7a (P = 0.08) suggest that Simmentals have a lesser ability to absorb and utilize dietary Cu, and may explain why Simmentals are more prone to Cu deficiency than Angus. Up-regulation of fetal liver Atox1, Cox17, and Commd1 in-Cu fetuses may reflect the great Cu demand by the fetus.

Effect of dietary boron on physiological responses in growing steers inoculated with bovine herpesvirus type-1

Thirty-six Angus and Angus×Simmental steers were fed one of three dietary treatments; (1) control (no supplemental B), (2) 5 mg supplemental B/kg, and (3) 15 mg supplemental B/kg for 47 days to determine the effects of dietary boron (B) on disease resistance following an inoculation with bovine herpesvirus type-1 (BHV-1). On day 34 of the study steers were inoculated intranasally with BHV-1. Rectal temperatures began to elevate at day 2, and plasma tumor necrosis factor-α concentrations increased (P<0.05) by day 2 following BHV-1 inoculation. Plasma acute phase proteins were increased (P<0.01) while plasma interferon-γ was decreased (P<0.05) by day 4 post-inoculation. Supplementation of B increased (P<0.001) plasma B concentrations in a dose-responsive manner. However, dietary B did not affect the duration and severity of clinical signs of BHV-1 and had minimal effects on plasma acute phase proteins and cytokines.

Effect of dietary copper amount and source on copper metabolism and oxidative stress of weanling pigs in short-term feeding.

Forty-eight weanling barrows were used to determine the effects of amount and source of dietary Cu on Cu metabolism, oxidative stress in the duodenum, and VFA ratios in the cecum of weanling pigs in short-term feeding. At 21 d of age, newly weaned pigs were stratified by BW (7.03 ± 1.20 kg) and equally assigned to 1 of the following dietary treatments: 1) control (5 mg supplemental Cu/kg diet from CuSO4), 2) 225 mg supplemental Cu/kg diet from CuSO4, or 3) 225 mg supplemental Cu/kg diet from tribasic Cu chloride (TBCC). Pigs were housed 2 pigs per pen and were fed a complex diet until harvest on d 11 and 12. During harvest, bile and liver were obtained for mineral analysis, and liver samples were obtained for analysis of mRNA expression of Cu regulatory proteins. Digesta of duodenum, proximal jejunum, and ileum were collected for soluble Cu analysis. Mucosal scrapings of duodenum, proximal jejunum, and ileum were obtained for analysis of mucosal Cu concentration and mRNA expression of Cu regulatory proteins. Duodenal mucosal scrapings were also collected for analysis of malondialdehyde (MDA). Pigs fed high Cu had markedly greater (P < 0.0001) Cu concentrations in the duodenal, proximal jejunal, and ileal mucosa than controls. Copper in the duodenal mucosa was greater (P = 0.003) in CuSO4 than TBCC pigs. Duodenal MDA concentrations were greater (P = 0.003) in CuSO4 vs. control pigs and tended (P = 0.06) to be greater than in TBCC pigs. Duodenal antioxidant 1 (Atox1) mRNA was downregulated (P < 0.01) in pigs fed high Cu compared to controls and was not affected by Cu source. Compared with control pigs, those fed CuSO4 and TBCC had greater (P < 0.001) liver and bile Cu concentrations. Liver Cu was also greater (P = 0.0007) in TBCC than CuSO4-fed pigs. Hepatic Cu transporting β-polypeptide ATPase (Atp7b) was upregulated (P = 0.02) in the Cu-supplemented pigs compared with controls and did not differ among Cu sources. The acetate:propionate ratio in cecal contents was much greater in pigs supplemented with 225 mg Cu/kg diet than in controls. When fed at 225 mg Cu/kg diet, TBCC may cause less oxidative stress in the duodenum than CuSO4. Feeding weanling pigs increased Cu resulted in modulation of duodenal and liver at the transcription level.