Inflammation markers predict zinc transporter gene expression in women with type 2 diabetes mellitus

Available online at www.sciencedirect.com

Journal of Nutritional Biochemistry xx (2013) xxx – xxx

Inflammation markers predict zinc transporter gene expression in women with type 2 diabetes mellitus☆,☆☆,★ Meika Foster a , Peter Petocz b , Samir Samman a,⁎ a

Discipline of Nutrition and Metabolism, School of Molecular Bioscience, University of Sydney NSW 2006, Australia b Department of Statistics, Macquarie University, NSW 2109, Australia

Received 23 July 2012; received in revised form 30 January 2013; accepted 10 February 2013

Abstract The pathology of type 2 diabetes mellitus (DM) often is associated with underlying states of conditioned zinc deficiency and chronic inflammation. Zinc and omega-3 polyunsaturated fatty acids each exhibit anti-inflammatory effects and may be of therapeutic benefit in the disease. The present randomized, doubleblind, placebo-controlled, 12-week trial was designed to investigate the effects of zinc (40 mg/day) and α-linolenic acid (ALA; 2 g/day flaxseed oil) supplementation on markers of inflammation [interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, C-reactive protein (CRP)] and zinc transporter and metallothionein gene expression in 48 postmenopausal women with type 2 DM. No significant effects of zinc or ALA supplementation were observed on inflammatory marker concentrations or fold change in zinc transporter and metallothionein gene expression. Significant increases in plasma zinc concentrations were observed over time in the groups supplemented with zinc alone or combined with ALA (P=.007 and P=.009, respectively). An impact of zinc treatment on zinc transporter gene expression was found; ZnT5 was positively correlated with Zip3 mRNA (Pb.001) only in participants receiving zinc, while zinc supplementation abolished the relationship between ZnT5 and Zip10. IL-6 predicted the expression levels and CRP predicted the fold change of the ZnT5, ZnT7, Zip1, Zip7 and Zip10 mRNA cluster (Pb.001 and P=.031, respectively). Fold change in the expression of metallothionein mRNA was predicted by TNF-α (P=.022). Associations among inflammatory cytokines and zinc transporter and metallothionein gene expression support an interrelationship between zinc homeostasis and inflammation in type 2 DM. © 2013 Elsevier Inc. All rights reserved. Keywords: Zinc transporter; Inflammation; Gene expression; Type 2 diabetes mellitus; Randomized controlled trial

1. Introduction There is increasing evidence that a state of nonresolving lowgrade inflammation is involved in the pathology of type 2 diabetes mellitus (DM). The aberrant expression of a range of immune mediators, such as C-reactive protein (CRP) and the inflammatory cytokines interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-α, often is reported in the disorder. The potential for dietary Abbreviations: ALA, α-linolenic acid; CPT, cell preparation tube; CP, crossing point; CRP, C-reactive protein; DM, diabetes mellitus; MT, metallothionein; n-3, omega-3; PBMC, peripheral blood mononuclear cells; rRNA, ribosomal ribonucleic acid; SLC, solute carrier; Zip, Zrt- and Irt-like protein; ZnT, zinc transporter. ☆ Financial support provided by The Medical Advances Without Animals (MAWA) Trust and Sydnovate, The University of Sydney. ☆☆ Meika Foster, no conflicts of interest; Peter Petocz, no conflicts of interest; Samir Samman, no conflicts of interest. ★ Statement of authors' contributions to manuscript: M.F. and S.S. designed research; M.F. conducted research; M.F., S.S. and P.P. analyzed data; M.F. and S.S. wrote the paper and had responsibility for final content. All authors read and approved the final manuscript. ⁎ Corresponding author. Tel.: +61 2 9351 2476; fax: +61 2 9351 5858. E-mail address: [email protected] (S. Samman). 0955-2863/$ - see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jnutbio.2013.02.006

components to modulate inflammatory processes alludes to new approaches in the management of type 2 DM. Zinc and omega-3 polyunsaturated fatty acids (n-3 PUFA) each exhibit anti-inflammatory effects [1,2] and therefore may have therapeutic benefit in alleviating the chronic inflammatory state. Type 2 DM exhibits an impaired immune function as part of its pathogenesis that ultimately results in a decreased functional β-cell mass [3]. A prospective examination of the effects of IL-1β, IL-6 and TNF-α on the development of type 2 DM found that participants with detectable levels of IL-1β and elevated levels of IL-6 in plasma had a threefold increased risk of developing DM compared to the reference group [4]. A large prospective study recently concluded that higher zinc intakes may be associated with a lower risk of type 2 DM in women [5]. Zinc is crucial for the normal development and function of cells, mediating both innate and acquired immunity [6]. In its signaling capacity, available zinc has the ability to regulate both negatively and positively the secretion of inflammatory cytokines, suggesting a potential interaction between the impaired immunity in type 2 DM and perturbed cellular zinc homeostasis [1]. Zinc concentrations in mammalian cells are maintained by two classes of zinc transporters: the ZnT [solute carrier (SLC)30] and Zrtand Irt-like protein (Zip) (SLC39) zinc transporter families. Metallothionein (MT) also is believed to play a central role in the

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maintenance of zinc homeostasis by trafficking zinc through the cell and releasing it to zinc-requiring proteins. Inflammation has been associated with modulated zinc transporter [7–10] and MT [11,12] expression in a variety of cell types. In turn, zinc transporters have been shown to be integral to cytokine production [13]. The potential interrelationship between zinc homeostasis and inflammation suggests that zinc supplementation may be of use in the treatment of type 2 DM. The anti-inflammatory effects of zinc include the ability to attenuate CRP and inflammatory cytokine production [1]. The effect of zinc on inflammatory markers has been explored in a number of human studies. In intervention trials measuring the effects of zinc on cytokine production in primary human blood cells, zinc supplementation was shown to reduce circulating levels of CRP and IL-6 in healthy older adults [14] and to decrease ex vivo generated levels of inflammatory cytokine mRNA and proteins in stimulated mononuclear cells [15–17]. Conversely, increased cytokine levels have been shown in stimulated mononuclear cells isolated from populations supplemented with ≤ 20 mg zinc/day [18–20], suggesting a dose effect of zinc. Few studies have explored the potential for zinc to ameliorate markers of inflammation in populations with type 2 DM. As with zinc, the potential for n-3 PUFA to influence inflammatory processes has been the focus of much research interest. In prospective epidemiological studies, long-chain n-3 PUFA in plasma and erythrocyte membrane are correlated inversely with concentrations of plasma inflammatory markers, including CRP and IL-6 [21,22]. These observations are supported by intervention trials that demonstrate a reduction in circulating CRP levels in subjects with metabolic syndrome supplemented with eicosapentaenoic acid (EPA) [23] and a decrease in CRP and IL-6 in overweight women receiving combined EPA and docosahexaenoic acid (DHA) supplementation [24]. A diet high in alpha-linolenic acid (ALA) has been shown to inhibit the mononuclear cell production of IL-6, IL-1β and TNF-α and decrease serum TNF-α [25] and CRP concentrations [26] in hypercholesterolemic subjects. The effects of ALA on inflammatory marker concentrations in type 2 DM are as yet undefined. The present study aims to investigate the effects of zinc and ALA supplementation on circulating concentrations of inflammatory markers (IL-1β, IL-6, TNF-α, CRP) and zinc transporter and MT gene expression in postmenopausal women with type 2 DM.

2.2. Randomization and intervention The study was a randomized, double-blind, placebo-controlled trial conducted over 12 weeks. Participants, the trial coordinator and personnel, and outcome investigators were blinded to treatment allocation. Upon enrolment, eligible participants (n=48) were randomized into four equal groups according to a computer-generated random-number sequence to receive a total of 40 mg/day elemental zinc (‘Zn group’), 2000 mg/day flaxseed oil (‘ALA group’), both zinc and flaxseed oil (‘Zn+ALA group’) or placebo. The zinc dose of 40 mg/day was selected as it represents the upper level of recommended zinc intake [28] and is similar to the median zinc dose (30 mg/day) used in a meta-analysis investigating the effects of zinc on plasma lipoprotein cholesterol concentrations in humans [29]. A supplement of 2000 mg flaxseed oil provides 1200 mg ALA; this amount was chosen to ensure that participants achieved the adequate intake of ALA, which is defined as 800 mg/day for adult women [28]. Study treatment was given as four capsules to be taken daily. In order to minimize the potential for side effects and enhance absorption in those receiving zinc [30,31], participants were instructed to consume the supplements in two equal parts (one part in the morning, one part in the evening, before food). Zinc and flaxseed oil capsules differed in appearance, and so each had a matching placebo. Zinc active and placebo capsules were prepared by a compounding pharmacist (Health Information Pharmacy, Balmain, Sydney, Australia). Each zinc active capsule comprised a clear shell containing 20 mg of elemental zinc (in the form of zinc sulphate monohydrate; Blackmores Ltd., Sydney, Australia) and cellulose filler as required, resulting in a capsule of white appearance; zinc placebo capsules contained cellulose and were identical in appearance to their active counterparts. ALA active capsules (Blackmores Ltd., Sydney, Australia) each comprised an opaque black shell containing 1000 mg of flaxseed oil (equivalent to 600 mg ALA). ALA placebo capsules (Catalent Australia Pty. Ltd., Victoria, Australia) utilized an identical opaque black shell filled with 1000 mg of olive oil. Olive oil was chosen as the placebo due to its low content of ALA. The fatty acid composition of the olive oil was as follows: palmitic acid 9.7%; stearic acid 2.5%; oleic acid 74.5%; linoleic acid 11.4%; ALA 0.7%. Quality control was conducted at the beginning of the study to verify supplement content. Capsules were packaged in 4-weekly lots (60 capsules); labeled with coded assignment numbers; and dispensed at baseline, week 4 and week 8. Compliance with the intervention was assessed by capsule counting at weeks 4, 8 and 12. A questionnaire was administered at each follow-up appointment to ascertain self-reported compliance and to determine whether participants were experiencing any positive or negative effects that they attributed to the supplements.

2.3. Collection of descriptive information Height (to ±0.1 cm) was measured at baseline with a wall-mounted stadiometer, and body weight (to ±100 g) was measured at each time point with an electronic calibrated scale. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Data on age, change in weight over the 3-month period preceding the study, time since DM diagnosis and current management strategies, family history of DM, usual alcohol consumption and use of nutritional supplements were selfreported at baseline.

2. Methods and materials 2.4. Collection of dietary information 2.1. Participants Women with type 2 DM were contacted and invited to participate in the trial via established mailing lists of DM organizations. In addition, the study was advertised in newspapers local to Sydney, Australia, through medical and dietetic practices, and around the University of Sydney campus. Women who indicated an interest in enrolling in the study were sent detailed information about the trial and an initial questionnaire to complete and return for the determination of participant eligibility. The primary inclusion criteria were that participants be postmenopausal women (no menses for N12 months) with type 2 DM (controlled by either diet and lifestyle or oral hypoglycemic medication prescribed within the previous 7 years) and have a normal glomerular filtration rate (N60 ml/min/1.73 m2) and microalbumin/creatine ratio (b3.5 mg/mmol). The requirement that participants be postmenopausal was intended to reduce confounding by estrogen fluctuations, which may influence zinc transporter mRNA expression [27]. Exclusion criteria were as follows: diagnosis with any current major illness other than DM, the use of insulin, tobacco use and the taking of prescription medications known to interact with zinc (e.g., medications for depression). The use of medications for the treatment of diabetic comorbidities [such as hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) for hypercholesterolemia and angiotensin-converting enzyme inhibitors for hypertension] was not an exclusion criterion. Potential participants were advised that they would be expected to refrain from giving blood donations and from the use of all nutritional supplements (excluding trial supplements) in the 6 weeks prior to the trial and throughout the trial period. The Human Research Ethics Committee of the University of Sydney approved the study protocol, and all participants provided written informed consent. The protocol was registered at www.clinicaltrials.gov (NCT01505803).

Dietary intake information was collected at baseline and at week 12 to correspond with the analyses of zinc transporter gene expression levels. Participants completed two nonsequential estimated food records (one weekday record and one weekend day record) in the 7-day period prior to their participation in the study for the assessment of baseline dietary intake. This process was repeated in the 7-day period prior to the final blood collection appointment for the assessment of week 12 dietary intake. Participants were instructed as to the appropriate days on which to record their food intake and provided with food record templates and instructions on how to complete them [32]. The research dietitian checked each record in the presence of the participant for consistency and completeness. Food records were analyzed (Foodworks, Professional Edition 2009; Xyris Software, QLD, Australia) to determine energy, protein, fat [total, saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), PUFA], carbohydrate, dietary fiber, alcohol and zinc intakes. The results from the two food records at each time point were averaged to obtain the final baseline and week 12 nutrient intake values.

2.5. Blood collection Blood collection procedures were in accordance with the guidelines of the International Zinc Nutrition Consultative Group [33]. Venous blood samples were collected at each time point from participants in the seated position after an overnight fast of at least 10 h. Mononuclear cell preparation Vacutainer tubes [cell preparation tube (CPT) Vacutainer; Becton Dickinson] were used for the isolation of peripheral blood mononuclear cells (PBMC), serum gel tubes for cytokine and CRP analyses, and trace metal tubes (Becton Dickinson) for plasma zinc analysis.

M. Foster et al. / Journal of Nutritional Biochemistry xx (2013) xxx–xxx

2.6. Zinc transporter and metallothionein gene expression All CPT Vacutainer tubes were centrifuged within 2 h of collection, and samples were immediately processed through to cDNA and stored at −80°C. Quantitative realtime polymerase chain reaction (PCR) analysis was conducted within 7 days of sample collection. The method for measuring zinc transporter and MT mRNA expression has been described previously [34]. In brief, PBMC from individual samples were extracted, and total RNA was prepared using the RNAqueous Small Scale Phenol-Free Total RNA Isolation Kit (Applied Biosystems-Life Technologies Australia Pty. Ltd., Victoria, Australia) according to the manufacturer's instructions. Total RNA was reverse transcribed into cDNA using the Superscript VILO cDNA Synthesis System (Invitrogen-Life Technologies Australia Pty. Ltd., Victoria, Australia) following the manufacturer's protocol. Relative quantification of zinc transporter mRNA was conducted using Taqman real-time PCR (ABI 7500 Fast Sequence Detection System; Applied BiosystemsLife Technologies Australia Pty. Ltd., Victoria, Australia). Inventoried Taqman gene expression assays and one custom-designed assay were obtained for ZnT1, ZnT5, ZnT6, ZnT7, ZnT8, Zip1, Zip3, Zip7, Zip10, MT-1A and MT-2A mRNA; the endogenous reference sequence used was 18S ribosomal ribonucleic acid (rRNA) (Applied Biosystems-Life Technologies Australia Pty. Ltd., Victoria, Australia; Supplemental table 1). The zinc transporters were selected to canvass import and export functions across both the plasma and intracellular membranes and/or because they had been shown to be zinc responsive in previous studies in humans, animal models and cell culture systems [27]. The amplification efficiency of each assay was determined using the standard curve method [35]. Amplification was conducted over 40 cycles, with each cycle comprising a denaturing (15 s at 95°C) and an annealing/extending (60 s at 60°C) step. Messenger RNA expression levels were normalized to 18S rRNA expression as an endogenous reference [36] and quantified using the ΔCP method; fold change was quantified using the ΔΔCt method [37]. 2.7. Cytokines and CRP Serum for cytokine analysis was obtained after centrifugation (3000 rpm, 15 min, 4°C) and stored at −80°C for subsequent analysis. The human cytokine/chemokine Milliplex MAP kit (Millipore, Billerica, MA, USA) was used for the simultaneous quantification of serum IL-1β, IL-6 and TNF-α concentrations according to the manufacturer's instructions. Samples were analyzed on a Luminex 100 Bioanalyzer (Luminex Corp., Austin, TX, USA) using Fidis multiplex technology (Biomedical Diagnostics, Marne la Vallée, France). Cytokine concentrations (pg/ml) were quantified using the median fluorescence intensity compared against a standard curve of known protein concentrations (Luminex Corp., Austin, TX, USA). Human cytokine standard, control and limit of detection values were included with each reaction plate. The measuring range for each assay was 0–10,000 pg/ml. Intra- and interassay precisions were, respectively, 6% and 7% for IL-1β, 8% and 12% for IL-6 and 11% and 16% for TNF-α. CRP was measured using the Tina-quant CRP (gen.3) immunoturbidimetric method adapted for a Roche Modular PPE analyzer (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. The measuring range was 0.3–350 mg/L. Inter- and intraassay variabilities were b10%. 2.8. Plasma zinc Plasma from trace metal tubes was prepared by centrifugation at 4°C for 10 min at 3000 rpm and stored at −80°C until further analysis. Plasma zinc was determined using inductively coupled plasma mass spectrometry (Agilent 7500ce ICPMS, Santa Clara, CA, USA). Samples were diluted (1:40) in ammonium EDTA using rhodium as an internal standard and measured against a matrix-matched standard curve prepared in the same dilution. Samples were analyzed in duplicate in a single batch (coefficient of variation b 5%), and appropriate quality control was utilized (Trace Element Control Serum, UTAK Laboratories, Inc, Valencia, CA, USA). 2.9. Sample size calculation Sample size estimations for the study assumed a standard deviation (S.D.) of change that was 20% of the mean baseline value for TNF-α. In order to detect a 20% difference in the TNF-α concentration from baseline in each group with 80% power and 5% significance, it was projected that 10 participants per treatment arm would be required. The planned enrollment of 48 participants allowed for a 20% attrition rate. The sample size calculations were conducted using Minitab version 16 (www.minitab. com).

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of all participants who received zinc supplements with all participants who were not supplemented with zinc. Bivariate correlations between zinc transporter and MT gene expression levels were calculated using the Pearson correlation coefficient. Comparisons between participants who expressed ZnT8 mRNA and participants in whom ZnT8 mRNA was not expressed were conducted using unpaired Student’s t tests. Principal component analysis was used to identify ‘clusters’ of zinc transporter and MT gene expression. Multivariate ANOVA models were used to determine whether the inflammatory markers or plasma zinc predicted each cluster. Linear regression models were constructed to explore associations between plasma zinc values and zinc transporter and MT mRNA expression levels, dietary zinc intake, age and BMI. BMI was included in all analyses as a covariate. The residuals from the regression models were checked to see if they satisfied the assumptions of normality and homoscedasticity: the initial regressions indicated that analyses of MT-1A and MT-2A were more appropriately conducted on a log scale. Statistical analyses were carried out using SPSS (PASW) version 18 (SPSS Inc, www.spss.com). A value of Pb.01 was taken to designate statistical significance. A conservative approach was taken such that Pb.05 was interpreted as statistically marginal due to the large number of test results under investigation.

3. Results Of 306 potential participants who indicated interest in enrolling in the study, 87 returned eligibility questionnaires. Twenty-two women did not meet the eligibility criteria, 12 women declined to participate despite being eligible, and 5 others withdrew their interest due to unexpected caregiver responsibilities or other family-related reasons. Forty-eight women were enrolled in the trial. Supplemental figure 1 shows the trial profile. Forty-three participants completed the trial and were included in the statistical analysis. A ‘complete case’ analysis was preferred to a strict intention-to-treat approach as only baseline data were available for four of the five participants lost to follow-up, making it difficult to justify the imputation of missing data for weeks 4, 8 and 12. The frequency and causes of participant withdrawal from the trial did not relate to treatment. The age and BMI of the 43 participants who completed the trial were, respectively, (mean±S.D.) 65.0±7.8 years and 28.6±5.1 kg/m2. The random glucose concentration and hemoglobin A1c of participants (n=43) at baseline were 6.9±0.3 mmol/L (mean±S.E.) and 6.7%±0.1%, respectively. The average time since diagnosis of type 2 DM was 6.5±5.2 years. Participants reported taking oral hypoglycemic (63%), lipid-lowering (72%) and other (67%) prescription medication. Three participants did not take any medication. There were no significant differences in the baseline characteristics or dietary intakes of participants after randomization into intervention groups; however, the proportion of participants who reported taking oral hypoglycemic agents tended to be lower in the ALA group (P= .07) (Supplemental table 2). There were no significant differences between baseline and week 12 dietary intakes of energy, protein, total fat, SFA, MUFA, carbohydrate, alcohol and zinc in participants who completed the trial when analyzed overall or after randomization into intervention groups; dietary fiber was marginally lower at week 12 compared to baseline in all participants (27.6±11.6 vs 24.2±9.3 g/ day, Pb.05) and in the placebo group (27.6±12.2 vs 22.1±9.4 g/day, Pb.05), and PUFA was marginally higher at week 12 compared to baseline in the ALA group (9.0±5.1 vs 11.9±6.4 g/day, Pb.05). Overall compliance with the interventions (based on capsule counting and participant self-reporting) was 94.1%±5.6%, with no significant difference between groups.

2.10. Statistics Differences in group means at baseline were investigated using analysis of variance (ANOVA) for continuous data and the Pearson χ2 test for categorical variables. Repeated-measures ANOVA of each factor by treatment group was undertaken, with a Greenhouse–Geisser adjustment for asphericity. Differences in the concentrations of biochemical measures over time within each treatment group were assessed using multivariate analysis. The significance of the change in the mean value for each variable (week 12−baseline) was calculated within each group using paired Student’s t tests. Post hoc investigations using Student’s t tests were undertaken to compare the results

3.1. Inflammatory markers and plasma zinc The concentrations of IL-1β, IL-6, TNF-α and CRP in participants (n=43) at baseline were (mean±S.E.) 0.92±0.28 pg/ml, 1.59±0.29 pg/ml, 10.4±0.6 pg/ml and 3.0±1.2 mg/L, respectively; the ranges were 0–7.6 pg/ml (IL-1β), 0–10.0 pg/ml (IL-6), 5.0–18.9 pg/ml (TNF-α) and 0.3–49.3 mg/L (CRP). Using repeated-measures

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Table 1 Effects of zinc (n=12), ALA (n=10) and zinc+ALA (n=11) interventions compared to placebo (n=10) over time (weeks 4, 8 and 12 compared to baseline) on serum cytokines (pg/ ml), CRP (mg/L) and plasma zinc (μmol/L) in participants who completed the trial Factor, group

P (RM) a

IL-1β (pg/ml) Zn ALA Zn+ALA Placebo IL-6 (pg/ml) Zn ALA Zn+ALA Placebo TNF-α (pg/ml) Zn ALA Zn+ALA Placebo CRP (mg/L) Zn ALA Zn+ALA Placebo Zinc (μmol/L) Zn ALA Zn+ALA Placebo

.62

Baseline

Week 4

Week 8

Week 12

Pb

Change c

Pd

1.03±0.55 0.39±0.60 1.04±0.57 1.19±0.60

1.15±0.60 0.47±0.66 0.86±0.63 0.37±0.66

0.86±0.47 0.00±0.51 0.26±0.49 0.74±0.51

1.13±1.00 0.33±1.10 0.78±1.05 1.86±1.10

.98 .44 .51 .56

0.11±0.47 −0.06±0.26 −0.25±0.60 0.67±1.17

.82 .82 .68 .58

1.88±0.56 1.43±0.62 1.60±0.59 1.39±0.62

1.90±0.63 1.69±0.69 1.07±0.66 1.52±0.69

1.88±0.61 0.98±0.67 1.92±0.64 0.78±0.67

0.93±0.36 1.43±0.39 0.99±0.37 1.07±0.39

.33 .44 .62 .16

−0.96±0.68 0.00±0.29 −0.61±0.51 −0.32±0.27

.18 1.00 .26 .27

10.3±1.1 10.9±1.2 9.7±1.1 10.6±1.2

9.4±1.0 9.2±1.1 9.6±1.0 8.7±1.1

9.5±0.8 10.1±0.9 9.3±0.8 8.0±0.9

9.3±1.0 10.0±1.1 8.7±1.1 8.6±1.1

.31 .64 .55 .06

−1.1±0.9 −0.9±1.2 −1.0±1.6 −2.0±0.9

.24 .45 .53 .054

1.2±0.8 2.5±0.9 3.0±0.8 2.0±0.9

1.2±1.0 3.5±1.1 3.1±1.0 2.2±1.1

1.5±0.8 2.7±0.8 2.7±0.8 1.5±0.8

0.9±0.7 3.4±0.7 2.4±0.7 1.9±0.7

.50 .65 .24 .10

−0.3±0.1 0.9±0.8 −0.6±0.5 −0.1±0.1

.07 .30 .31 .48

13.4±0.5 11.7±0.6 12.6±0.6 13.3±0.6

15.3±0.7 12.3±0.8 14.5±0.8 12.8±0.8

15.3±0.5 12.3±0.6 15.2±0.6 12.6±0.6

16.3±0.5 12.8±0.5 15.0±0.5 13.4±0.5

.007 .11 .009 .40

.77

.93

.71

.056 2.8±0.7 1.1±0.6 2.4±0.8 0.1±0.4

.002 .10 .019 .81

Data are expressed as mean±S.E. There were no significant differences in biochemical measures among intervention groups at baseline. Reference intervals: CRP ≤5 mg/L; plasma zinc, 10–18 μmol/L. a Repeated-measures analysis (factor*group P value with Greenhouse–Geisser adjustment for asphericity). b Differences over time (within each group). c Week 12 minus baseline values. d P value for change (t test within each group).

ANOVA, no significant effects of zinc and ALA supplementation, administered alone or in combination, on serum concentrations of inflammatory markers were observed (Table 1). The plasma zinc concentration of participants (n=43) at baseline was 12.8±0.3 μmol/L (mean±S.E.). In all four intervention groups,

plasma zinc values were within the reference range (10–18 μmol/L), and there were no significant differences between groups at baseline. Significant increases in plasma zinc were observed over time in the Zn and Zn+ALA groups (P=.007 and P=.009, respectively), but not in the groups supplemented with ALA or

Fig. 1. Box plot representations of zinc transporter and metallothionein mRNA fold change in PBMC of postmenopausal women with type 2 DM according to whether they received (n=22) or did not receive (n=18) 40 mg/day zinc sulphate for 12 weeks. Data are calculated using ΔΔCt and expressed on a log scale.

M. Foster et al. / Journal of Nutritional Biochemistry xx (2013) xxx–xxx

Fig. 2. Relationship between ZnT5 and Zip3 mRNA in PBMC of women with type 2 DM after zinc supplementation for 12 weeks (n=22). Values are zinc transporter mRNA/ 106 18S rRNA.

placebo. In the Zn group, the difference in the plasma zinc concentration at week 12 compared to baseline was +2.8 μmol/L (P=.002); after Zn+ALA supplementation, the increase was +2.4 μmol/L (P=.019) (Table 1). In a regression analysis, plasma zinc concentrations at baseline were predicted by MT-2A mRNA expression levels (P=.001) (data not shown). 3.2. Zinc transporter and metallothionein expression At baseline, ZnT1, ZnT7, Zip1 and MT-2A mRNA were expressed at levels approximately one- to threefold higher than ZnT5, ZnT6, Zip3, Zip7, Zip10 and MT-1A (Supplemental figure 2). ZnT8 mRNA expression was of low abundance and detected in 21 of the 40 participants. Compared to participants in whom ZnT8 mRNA was not expressed (n=19), participants who expressed ZnT8 mRNA (n=21) were observed at baseline to have lower ZnT5 mRNA expression levels (P=.041) and MUFA intakes (18.4 vs. 29.7 g/day; P=.002); in addition, ZnT8 mRNA expression was positively associated with TNF-α levels (r=0.48, P=.029; Supplemental figure 3).

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There were no mean differences between groups in zinc transporter and MT mRNA fold change over 12 weeks in response to Zn, ALA or Zn+ALA supplementation. When groups were combined according to whether participants received (n=22) or did not receive (n=18) zinc as part of their intervention, no significant up- or down-regulation of gene expression in response to zinc supplementation was observed (Fig. 1). A range of bivariate correlations in zinc transporter and MT gene expression was found. In particular, ZnT7, the most highly expressed zinc transporter mRNA, was positively associated in all participants at baseline with ZnT5, Zip1, Zip7 and Zip10 (Pb.001) and ZnT1, ZnT6, Zip3 and MT-2A (P≤0.01; Supplemental table 3). Coordination also was observed in zinc transporter mRNA fold change after 12 weeks. Significant positive relationships (Pb.002) were demonstrated for all bivariate combinations of ZnT1, ZnT5, ZnT6, ZnT7, Zip1, Zip3, Zip7 and Zip10 mRNA fold change. Relationships (.05NPb.001) were observed between the fold change of MT-2A and all other mRNA (data not shown). A number of relationships were observed that were affected by zinc treatment. ZnT5 mRNA was positively correlated at week 12 with Zip3 only in participants supplemented with zinc (r=0.66, Pb.001, n=22; Fig. 2). Other associations appeared to be abolished by zinc supplementation. A positive relationship between the mRNA expression levels of ZnT5 and Zip10 observed in participants who did not receive zinc (r=0.64, P=.004, n=18) was absent in those who were administered zinc supplements (r=0.08, P=.72, n=22). The relationships of ZnT6 and Zip7 with other transporters followed a similar pattern (Table 2); correlations observed at week 12 between ZnT6 mRNA and ZnT8, Zip1, Zip3 and Zip7 (Fig. 3) and between Zip7 mRNA and ZnT8, Zip1 and Zip3 were not seen in participants supplemented with zinc. Likewise, MT-2A fold change was correlated with the fold change of ZnT1 (r=0.67, P=.002) and Zip1 (r=0.65, P=.004) only in those not supplemented with zinc. 3.3. Relationships among zinc transporter and MT mRNA, plasma zinc and inflammatory markers Principal component analysis revealed three clusters each containing zinc transporters or MT mRNA that behaved in a similar

Table 2 Bivariate Pearson correlations between zinc transporter mRNA when analyzed according to whether participants received (n=22) or did not receive (n=18) zinc supplements for 12 weeks.

ZnT1 ZnT5 ZnT6 ZnT7 ZnT8 Zip1 Zip3 Zip7 Zip10 MT-1A a

Zinc No zinc Zinc No zinc Zinc No zinc Zinc No zinc Zinc No zinc Zinc No zinc Zinc No zinc Zinc No zinc Zinc No zinc Zinc No zinc

a

ZnT5

ZnT6

ZnT7

ZnT8

0.39 0.43

0.63 ⁎⁎ 0.57 ⁎ 0.59 ⁎⁎ 0.58 ⁎

0.63 ⁎⁎ 0.60 ⁎⁎ 0.74 ⁎⁎⁎ 0.81 ⁎⁎⁎ 0.50 ⁎ 0.57 ⁎

−0.87 0.81 ⁎⁎⁎ −0.26 0.13 0.09 0.63 ⁎⁎ −0.08 0.28

Zip1 0.48 ⁎ 0.59 ⁎ 0.53 ⁎ 0.53 ⁎ 0.52 ⁎ 0.59 ⁎ 0.74 ⁎⁎⁎ 0.56 ⁎ 0.01 0.36

Zip3 0.45 ⁎ 0.63 ⁎⁎ 0.66 ⁎⁎⁎ 0.42 0.34 0.87 ⁎⁎⁎ 0.46 ⁎ 0.36 −0.38 0.72 ⁎⁎⁎ 0.10 0.52 ⁎

Zip7

Zip10

MT-1A

0.46 ⁎ 0.56 ⁎ 0.29 0.47 0.22 0.91 ⁎⁎⁎ 0.51 ⁎ 0.44 0.04 0.66 ⁎⁎

0.46 ⁎ 0.46 0.08 0.64 ⁎⁎

0.07 0.39 0.27 0.36 −0.02 0.52 ⁎ 0.22 0.38 −0.28 0.39 −0.10 0.37 0.38 0.44 0.42 0.41 0.19 0.18

0.33 0.61 ⁎⁎ 0.29 0.78 ⁎⁎⁎

ZnT8 mRNA expression was detected in 11 participants supplemented with zinc and 10 participants not supplemented with zinc. ⁎ Pb.05. ⁎⁎ Pb.01. ⁎⁎⁎ Pb.001.

0.19 0.63 ⁎⁎ 0.41 0.60 ⁎⁎ 0.36 0.27 0.17 0.42 0.12 0.45 0.59 ⁎⁎ 0.58 ⁎

MT-2A 0.43 ⁎ 0.55 ⁎ 0.45 ⁎ 0.64 ⁎⁎ 0.10 0.65 ⁎⁎ 0.65 ⁎⁎ 0.65 ⁎⁎ −0.18 0.41 0.38 0.49 ⁎ 0.29 0.49 ⁎ 0.55 ⁎⁎ 0.60 ⁎⁎ 0.30 0.38 0.66 ⁎⁎⁎ 0.83 ⁎⁎⁎

6

M. Foster et al. / Journal of Nutritional Biochemistry xx (2013) xxx–xxx

Fig. 3. Relationship between Zip7 and ZnT6 mRNA at week 12 in PBMC of women with type 2 DM who did not receive zinc supplementation (n=18). Values are zinc transporter mRNA/106 18S rRNA.

manner. The clusters were identified as follows: cluster 1: {ZnT5, ZnT7, Zip1, Zip7 and Zip10}, cluster 2: {ZnT1, ZnT6, ZnT8 and Zip3} and cluster 3: {MT1A and MT2a}. A multivariate ANOVA model was used to determine whether the inflammatory markers or plasma zinc predicted mRNA levels (model 1). Cluster 1 {ZnT5, ZnT7, Zip1, Zip7 and Zip10 mRNA} was predicted at baseline by IL-6 which was a significant positive predictor of all transporters within the cluster (Pb.001). Cluster 2 was not predicted by any inflammatory markers or plasma zinc, and cluster 3 {MT-1A and MT-2A mRNA} was predicted by TNF-α (P=.014) and plasma zinc (P=.014). (Supplemental table 4). A second model (model 2) was used to investigate whether fold changes in the expression of zinc transporter and MT mRNA were predicted by the difference (week 12−baseline values) in concentrations of the inflammatory markers or plasma zinc. The first and second clusters were predicted by CRP (P=.031 and P=.018, respectively). The first cluster was predicted also by plasma zinc (P=.033). Cluster 3 was predicted by plasma zinc (P=.035) and TNF-α (P=.022) (Supplemental table 4). 4. Discussion No significant effects of zinc and ALA supplementation on inflammatory marker concentrations were observed in the present trial in postmenopausal women with type 2 DM. Previous investigations have reported inconsistent findings [38]. For instance, supplementation with 45 mg zinc/day for 6 months was found to reduce plasma CRP and IL-6 levels in men and women [14]. Likewise, 3 g/day DHA lowered circulating concentrations of CRP and IL-6 in hypertriglyceridemic men [39]. Conversely, no differences in CRP or markers of oxidative stress were observed in males with type 2 DM after supplementation with 240 mg zinc/day [40]. Supplementation with 4 g/day EPA or DHA did not influence plasma CRP, IL-6 or TNF-α levels in men and postmenopausal women with type 2 DM [41], and plasma inflammatory marker concentrations in adults with metabolic syndrome were not affected by plant- or marine-derived n-3 PUFA [42]. The underlying effects of medications prescribed in the treatment of type 2 DM and its cardiovascular disease risk factors on the inflammatory milieu may mask the anti-inflammatory effects of zinc and/or n-3 PUFA supplementation. The zinc transporter gene expression profile observed in the present study is comparable to our previous report in healthy men and women [34]; in both instances, ZnT1, ZnT7 and Zip1 were the most highly expressed zinc transporters, and the expression of ZnT8 was variable. The range of bivariate relationships observed among

zinc transporter and MT mRNA demonstrates that zinc transporter gene expression is coordinated in women with type 2 DM. Some of the associations appear to be influenced by zinc status, particularly those involving the mRNA of the intracellular zinc transporters ZnT5 and Zip7. ZnT5 mRNA was associated with Zip3 only in women who received zinc supplements, while zinc supplementation was found to abolish associations between ZnT5 and Zip10 mRNA. Participants who did not receive zinc supplements demonstrated relationships between Zip7 and the mRNA of ZnT6, Zip1, Zip3 and ZnT8. Consistent with these observations, Zip3 is reportedly present in intracellular organelles when zinc is replete [43], and transcriptional expression of Zip10 appears to be repressed under normal physiologic levels of zinc [44,45]. Zip7 appears to play a role in mobilizing zinc from the Golgi apparatus under zinc limiting conditions [46]. It would be useful for future studies to examine the content and distribution of intracellular zinc in conjunction with measurements of zinc transporter and MT expression to explore in more detail the effects of zinc supplementation on zinc homeostasis in this population. Up-regulation and down-regulation of zinc transporter and MT mRNA in primary blood cells after supplementation with zinc have been shown in healthy men [18,47,48] but were not apparent in the present trial. However, we observed that circulating baseline levels of IL-6 were able to predict the expression levels of a cluster of transporters (ZnT5, ZnT7, Zip1, Zip7 and Zip10 mRNA), such that each unit increase in IL-6 produced an 8%–15% increase in the expression of each zinc transporter gene. We hypothesize that the net result of IL-6-induced increases in this combination of zinc transporter mRNA is the redistribution of zinc into PBMC organelles as part of a coordinated inflammatory response. Our findings are supported by the results of others [9]. Inflammation-induced alterations in the expression profile of Zip genes in a model of allergic airway inflammation appeared to be directed toward increasing cellular zinc uptake which, the authors of the study suggest, enables the increased demand for zinc-dependent proteins under inflammatory conditions to be met [9]. The present study is novel in that little, if any, previous research has investigated the influence of zinc on zinc transporter and MT mRNA expression in women with type 2 DM. The sample size of the present trial may have been too small to detect changes in inflammatory markers, particularly given the potential for multiple etiologies in populations with type 2 DM [49] and the range of BMI values in our population. In summary, no effects of zinc and ALA supplementation on zinc transporter mRNA fold change and circulating levels of inflammatory markers were found in postmenopausal women with type 2 DM. The observations that IL-6 and CRP predict zinc transporter gene expression and TNF-α predicts MT mRNA fold change support a relationship in type 2 DM between inflammation and zinc homeostasis. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jnutbio.2013.02.006. References [1] Foster M, Samman S. Zinc and redox signaling: perturbations associated with cardiovascular disease and diabetes mellitus. Antioxid Redox Signal 2010;13: 1549–73, http://dx.doi.org/10.1089/ars.2010.3111. [2] Calder PC. Polyunsaturated fatty acids, inflammation, and immunity. Lipids 2001;36:1007–24. [3] Donath MY, Størling J, Maedler K, Mandrup-Poulsen T. Inflammatory mediators and islet beta-cell failure: a link between type 1 and type 2 diabetes. J Mol Med 2003;81:455–70. [4] Spranger J, Kroke A, Möhlig M, Hoffman K, Bergmann MM, Ristow M, Boeing H, Pfeiffer AF. Inflammatory cytokines and the risk to develop type 2 diabetes: results of the prospective population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam Study. Diabetes 2003;52:812–7. [5] Sun Q, van Dam RM, Willet WC, Hu FB. Prospective study of zinc intake and risk of type 2 diabetes in women. Diabetes Care 2009;32:629–34. [6] Ibs KH, Rink L. Zinc-altered immune function. J Nutr 2003;133:1452S–6S.

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