Jesse Bertinato

Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells

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.

Ctr1 transports silver into mammalian cells

Silver is a non-essential, toxic metal. The use of silver as an antimicrobial agent in many applications and its presence as a contaminant in foods and air can lead to accumulation in tissues. Despite its widespread use, the systems involved in the uptake of silver into mammalian cells are presently unknown. Previous studies have shown that copper uptake at the plasma membrane by copper transporter 1 (Ctr1) is inhibited by an excess of silver, suggesting that Ctr1 may function in importing silver into cells. In this study we examined directly the role of Ctr1 in the accumulation of silver in mammalian cells using over-expression experiments and mouse embryonic fibroblast cells lacking Ctr1. COS-7 cells transfected to express a human Ctr1-green fluorescent protein (hCtr1-GFP) fusion protein hyper-accumulated silver when incubated in medium supplemented with low micromolar concentrations (2.5-10 micromol/L) of AgNO(3). An hCtr1-GFPM150L,M154L variant deficient for copper transport failed to stimulate accumulation of silver. Mouse embryonic fibroblast cells lacking Ctr1 showed approximately a 50% reduction in silver content when incubated in silver-supplemented medium compared to a wild-type isogenic cell line. Collectively, these data demonstrate that Ctr1 transports both copper and silver and suggest that Ctr1 is an important transport protein in the accumulation of silver in mammalian cells.

Copper Chaperone for Cu/Zn Superoxide Dismutase is a sensitive biomarker of mild copper deficiency induced by moderately high intakes of zinc

BackgroundSmall increases in zinc (Zn) consumption above recommended amounts have been shown to reduce copper (Cu) status in experimental animals and humans. Recently, we have reported that copper chaperone for Cu/Zn superoxide dismutase (CCS) protein level is increased in tissues of overtly Cu-deficient rats and proposed CCS as a novel biomarker of Cu status.MethodsWeanling male Wistar rats were fed one of four diets normal in Cu and containing normal (30 mg Zn/kg diet) or moderately high (60, 120 or 240 mg Zn/kg diet) amounts of Zn for 5 weeks. To begin to examine the clinical relevance of CCS, we compared the sensitivity of CCS to mild Cu deficiency, induced by moderately high intakes of Zn, with conventional indices of Cu status.ResultsLiver and erythrocyte CCS expression was significantly (P < 0.05) increased in rats fed the Zn-60 and/or Zn-120 diet compared to rats fed normal levels of Zn (Zn-30). Erythrocyte CCS expression was the most sensitive measure of reduced Cu status and was able to detect a decrease in Cu nutriture in rats fed only twice the recommended amount of Zn. Liver, erythrocyte and white blood cell CCS expression showed a significant (P < 0.05) inverse correlation with plasma and liver Cu concentrations and caeruloplasmin activity. Unexpectedly, rats fed the highest level of Zn (Zn-240) showed overall better Cu status than rats fed a lower level of elevated Zn (Zn-120). Improved Cu status in these rats correlated with increased duodenal mRNA expression of several Zn-trafficking proteins (i.e. MT-1, ZnT-1, ZnT-2 and ZnT-4).ConclusionCollectively, these data show that CCS is a sensitive measure of Zn-induced mild Cu deficiency and demonstrate a dose-dependent biphasic response for reduced Cu status by moderately high intakes of Zn.

Increased incorporation of dietary plant sterols and cholesterol correlates with decreased expression of hepatic and intestinal Abcg5 and Abcg8 in diabetic BB rats.

The aim of this study was to determine the impact of dietary plant sterols and stanols on sterol incorporation and sterol-regulatory gene expression in insulin-treated diabetic rats and nondiabetic control rats. Diabetic BioBreeding (BB) and control BB rats were fed a control diet or a diet supplemented with plant sterols or plant stanols (5 g/kg diet) for 4 weeks. Expression of sterol-regulatory genes in the liver and intestine was assessed by real-time quantitative polymerase chain reaction. Diabetic rats demonstrated increased tissue accumulation of cholesterol and plant sterols and stanols compared to control rats. This increase in cholesterol and plant sterols and stanols was associated with a marked decrease in hepatic and intestinal Abcg5 (ATP-binding cassette transporter G5) and Abcg8 (ATP-binding cassette transporter G8) expressions in diabetic rats, as well as decreased mRNA levels of several other genes involved in sterol regulation. Plant sterol or plant stanol supplementation induced the accumulation of plant sterols and stanols in tissues in both rat strains, but induced a greater accumulation of plant sterols and stanols in diabetic rats than in control rats. Surprisingly, only dietary plant sterols decreased cholesterol levels in diabetic rats, whereas dietary plant stanols caused an increase in cholesterol levels in both diabetic and control rats. Therefore, lower expression levels of Abcg5/Abcg8 in diabetic rats may account for the increased accumulation of plant sterols and cholesterol in these rats.

Sparing effects of selenium and ascorbic acid on vitamin C and E in guinea pig tissues

BackgroundSelenium (Se), vitamin C and vitamin E function as antioxidants within the body. In this study, we investigated the effects of reduced dietary Se and L-ascorbic acid (AA) on vitamin C and α-tocopherol (AT) status in guinea pig tissues.MethodsMale Hartley guinea pigs were orally dosed with a marginal amount of AA and fed a diet deficient (Se-D/MC), marginal (Se-M/MC) or normal (Se-N/MC) in Se. An additional diet group (Se-N/NC) was fed normal Se and dosed with a normal amount of AA. Guinea pigs were killed after 5 or 12 weeks on the experimental diets at 24 and 48 hours post AA dosing.ResultsLiver Se-dependent glutathione peroxidase activity was decreased (P < 0.05) in guinea pigs fed Se or AA restricted diets. Plasma total glutathione concentrations were unaffected (P > 0.05) by reduction in dietary Se or AA. All tissues examined showed a decrease (P < 0.05) in AA content in Se-N/MC compared to Se-N/NC guinea pigs. Kidney, testis, muscle and spleen showed a decreasing trend (P < 0.05) in AA content with decreasing Se in the diet. Dehydroascorbic acid concentrations were decreased (P < 0.05) in several tissues with reduction in dietary Se (heart and spleen) or AA (liver, heart, kidney, muscle and spleen). At week 12, combined dietary restriction of Se and AA decreased AT concentrations in most tissues. In addition, restriction of Se (liver, heart and spleen) and AA (liver, kidney and spleen) separately also reduced AT in tissues.ConclusionTogether, these data demonstrate sparing effects of Se and AA on vitamin C and AT in guinea pig tissues.

Lower serum magnesium concentration is associated with diabetes, insulin resistance, and obesity in South Asian and white Canadian women but not men

Background A large proportion of adults in North America are not meeting recommended intakes for magnesium (Mg). Women and people of South Asian race may be at higher risk for Mg deficiency because of lower Mg intakes relative to requirements and increased susceptibility to diabetes, respectively. Objective This study compared serum Mg concentrations in South Asian (n=276) and white (n=315) Canadian women and men aged 20–79 years living in Canadas Capital Region and examined the relationship with diabetes, glucose control, insulin resistance, and body mass index. Results Serum Mg concentration was lower in women of both races and South Asians of both genders. Racial differences in serum Mg were not significant after controlling for use of diabetes medication. A substantial proportion of South Asian (18%) and white (9%) women had serum Mg <0.75 mmol/L indicating hypomagnesemia. Use of diabetes medication and indicators of poorer glucose control, insulin resistance, and obesity were associated with lower serum Mg in women, but not in men. Conclusions These results suggest that the higher incidence of diabetes in South Asians increases their risk for Mg deficiency and that health conditions that increase Mg requirements have a greater effect on Mg status in women than men.

Zinc Supplementation Does Not Alter Sensitive Biomarkers of Copper Status in Healthy Boys

The tolerable upper intake levels (UL) for zinc for children were based on limited data and there is concern that the UL may be set too low. The first effect of excessive zinc intake is a reduction in copper status. The primary objective of this study was to examine the effect of zinc supplementation on copper status in children. Healthy, 6- to 8-y-old boys from Ontario, Canada were assigned to take a placebo (n = 10) or 5 mg (n = 10), 10 mg (n = 9), or 15 mg (n = 8) of zinc supplement daily for 4 mo in a double-blinded, placebo-controlled, randomized trial. Biochemical measures were evaluated at baseline and after 2 and 4 mo of supplementation. Food records were completed near the baseline and 4-mo visits. Age and anthropometric measurements did not differ (P > 0.05) between treatment groups at baseline. Mean zinc intakes from food alone (10.9-14.8 mg zinc/d) approached or exceeded the UL of 12 mg/d. Compared with the placebo group, the zinc groups had a greater change in the urine zinc:creatinine ratio at 4 mo (P = 0.02). Traditional (plasma copper and ceruloplasmin activity) and more sensitive biomarkers of copper status, including erythrocyte SOD1 activity and the erythrocyte CCS:SOD1 protein ratio, were unchanged in zinc-supplemented boys, demonstrating that copper status was not depressed. Serum lipid measures and hemoglobin concentrations were also unaffected and gastrointestinal symptoms were not reported. These data provide evidence in support of the need for reexamining the current UL for zinc for children.

Small increases in dietary calcium above normal requirements exacerbate magnesium deficiency in rats fed a low magnesium diet

In North America, the calcium (Ca):magnesium (Mg) intake ratio has increased over the last several decades raising concerns about possible adverse effects of Ca intakes on Mg status. The primary objective of this study was to investigate whether small decreases or increases in dietary Ca from normal requirements worsen Mg status in rats fed a low Mg diet. Weanling male Sprague-Dawley rats were fed 1 of 8 diets for 6 weeks. The 7 test diets were supplemented with low Mg (0.18 g/kg diet) and either 1 (1Ca), 3 (3Ca), 5 (5Ca), 7.5 (7.5Ca), 10 (10Ca), 15 (15Ca) or 20 (20Ca) g Ca/kg diet. The control diet was supplemented with normal Mg (0.5 g/kg) and Ca (5 g/kg). Rats fed higher Ca gained less weight and had lower fat mass and energy efficiency. Compared to rats fed normal Ca (5Ca), Mg concentrations in serum and femur were lower in rats fed the higher Ca diets. Haemoglobin and haematocrit were also lower in rats fed the 15Ca and 20Ca diets. Rats fed the 10Ca, 15Ca and 20Ca diets had higher urine Ca compared to rats fed the 5Ca diet. Increase in urine Ca was associated with a rise in urine Mg. The higher Ca diets increased the Ca:Mg molar ratio in serum, femur, heart and kidney. These results suggest that small increases in dietary Ca exacerbate Mg deficiency in rats fed an inadequate Mg diet by reducing intestinal Mg absorption and also by impairing renal Mg reabsorption at higher Ca intakes.

Serum Magnesium Concentrations in the Canadian Population and Associations with Diabetes, Glycemic Regulation, and Insulin Resistance

Total serum magnesium (Mg) concentration (SMC) is commonly used to assess Mg status. This study reports current SMCs of Canadians and their associations with demographic factors, diabetes, and measures of glycemic control and insulin resistance using results from the Canadian Health Measures Survey cycle 3 (2012–2013). Associations were examined in adults aged 20–79 years using linear mixed models. Mean SMCs and percentile distributions for 11 sex-age groups between 3 and 79 years (n = 5561) are reported. SMCs were normally distributed and differences (p < 0.05) among sex and age groups were small. Between 9.5% and 16.6% of adult sex-age groups had a SMC below the lower cut-off of a population-based reference interval (0.75–0.955 mmol·L−1) established in the United States population as part of the NHANES I conducted in 1971–1974. Having diabetes was associated with 0.04 to 0.07 mmol·L−1 lower SMC compared to not having diabetes in the various models. Body mass index, glycated hemoglobin, serum glucose and insulin concentrations, and homeostatic model assessment of insulin resistance were negatively associated with SMC. This is the first study to report SMCs in a nationally representative sample of the Canadian population. A substantial proportion of Canadians are hypomagnesaemic in relation to a population-based reference interval, and SMC was negatively associated with diabetes and indices of glycemic control and insulin resistance.

Bioavailability of magnesium from inorganic and organic compounds is similar in rats fed a high phytic acid diet

A large section of the North American population is not meeting recommended intakes for magnesium (Mg). Supplementation and consumption of Mg-fortified foods are ways to increase intake. Currently, information on Mg bioavailability from different compounds and their efficacy in improving Mg status is scant. This study compared the relative ability of inorganic and organic Mg compounds to preserve the Mg status of rats when fed at amounts insufficient to retain optimal Mg status. Male Sprague-Dawley rats (n=12/diet group) were fed one of eight test diets supplemented with phytic acid (5 g/kg diet) and low levels of Mg (155 mg elemental Mg/kg diet) from Mg oxide, Mg sulphate, Mg chloride, Mg citrate, Mg gluconate, Mg orotate, Mg malate or ethylenediaminetetraacetic acid disodium Mg salt for five weeks. Rats were also fed three control diets that did not contain added phytic acid but were supplemented with 500 (NMgO, normal), 155 (LMgO, low) or 80 (DMgO, deficient) mg of Mg per kg diet as Mg oxide. Mg concentrations in femur, serum and urine showed a graded decrease in rats fed the control diets with lower Mg. Mg concentrations did not differ (P≥0.05) between rats fed the different test diets. Addition of phytic acid to the diet did not affect the Mg status of the rats. The results indicate that any differences in the Mg bioavailability of the compounds were small and physiologically irrelevant.