Demonstration of a Hyperglycemia-Driven Pathogenic Abnormality of Copper Homeostasis in Diabetes and Its Reversibility by Selective Chelatio
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糖尿病学杂志 2005年第5期
1 School of Biological Sciences, Faculty of Science, University of Auckland, New Zealand
2 Centre for Molecular Biodiscovery, Faculty of Science, University of Auckland, New Zealand
3 Department of Medicine, Faculty of Medical and Health Sciences, University of Auckland, New Zealand
4 Human Nutrition Unit, University of Auckland, New Zealand
5 South Auckland Diabetes Service, Middlemore Hospital, Otahuhu, New Zealand
6 School of Medical Sciences, Faculty of Medical and Health Sciences, University of Auckland, New Zealand
7 Department of Medicine, Christchurch Clinical School, University of Otago, Christchurch, New Zealand
ABSTRACT
We recently showed that treatment with the CuII-selective chelator, trientine, alleviates heart failure in diabetic rats, improves left ventricular hypertrophy in humans with type 2 diabetes, and increases urinary Cu excretion in both diabetic rats and humans compared with nondiabetic control subjects. In this study, we characterized the homeostasis of Cu and eight other nutritionally essential elements in diabetes under fully residential conditions in male subjects with type 2 diabetes and age-matched control subjects. We then probed elemental balance with oral trientine in a parallel-group, placebo-controlled study in these subjects. Before treatment, there were no detectable between-group differences in the balance of any element, although urinary output of several elements was greater in diabetic subjects. Mean extracellular superoxide dismutase (EC-SOD) activity was elevated in diabetic subjects, and its activity correlated strongly with the interaction between [Cu]serum and HbA1c. Trientine caused the Cu balance to become negative in diabetic subjects through elevated urinary Cu losses and suppressed elevated EC-SOD. Basal urinary Cu predicted urinary Cu losses during treatment, which caused extraction of systemic CuII. We suggest that cardiovascular complications in diabetes might be better controlled by therapeutic strategies that focus on lowering plasma glucose and loosely bound systemic CuII.
Oxidative stress has been implicated as a contributory mechanism in age-related disorders, including diabetes, hypertension, obesity, and atherosclerosis (1). Clinical trials of antioxidant (2) or carbonyl-trapping (3) agents in these disorders have had mixed success, indicating that the mechanisms underlying these disorders may be more complex than previously thought (3). In diabetes, the nature of oxidant stress and how it might promote complications is still unclear.
Free Fe and Cu ions are highly redox active (4) and might contribute to tissue damage by the generation of reactive oxygen species (ROS) (1), but the in vivo availability of catalytic Fe and Cu is usually very restricted (4). Both metals have been previously discussed in relation to the mechanisms of diabetes complications (5), but abnormalities of Fe homeostasis have not been linked to the major classes of diabetes and it is unknown whether altered Cu metabolism is relevant to diabetes complications.
Heart disease leads to death in most diabetic subjects (6,7). The Cu-selective chelator, trientine, causes a CuII-trientine complex to appear in the urine of diabetic rats whose abnormal Cu metabolism occurred after diabetes induction (8); trientine alleviated heart failure, improved cardiomyocyte structure, and reversed elevations in left ventricle collagen and 1 integrin without lowering blood glucose (8). Trientine also increased Cu excretion and decreased left ventricle mass in diabetic humans (8), implicating increased systemic CuII in the mechanism by which diabetes damages the heart.
Here we detail measurements of a 6-day balance of nine elements, including Cu and Fe, in diabetic and age-matched nondiabetic control subjects in whom we probed systemic metal balance with oral trientine in a subsequent study. Basal urinary output of Cu and Fe was significantly increased in diabetes, and those output values correlated strongly. Trientine treatment increased urinary excretion of Cu in a dosage-dependent manner, as predicted by basal urinary Cu, thereby causing a positive Cu balance to become negative in diabetes. In contrast, it modified neither Fe balance nor rates of urinary or fecal Fe excretion in these subjects. Furthermore, trientine did not render negative the balance of any other element in either diabetic or control subjects. Regulation of Cu metabolism was shown to be abnormal in diabetes and selectively modified by trientine, which did not concomitantly modify Fe metabolism. These findings are consistent with the effects of trientine in reversing the tendency to systemic accumulation of increased, loosely bound CuII in diabetes, which may help explain its therapeutic effects in diabetic cardiovascular disease.
RESEARCH DESIGN AND METHODS
Men (age 30eC70 years) with normal electrocardiograms were recruited for this study. Diabetic subjects had been diagnosed at least 6 months prior to the study, and control subjects had normal glucose tolerance. Exclusion criteria included the presence of type 1 diabetes; nephropathy; abnormal hematology or Fe deficiency; a history of significant cardiac disease; previous hepatic, gastrointestinal, or endocrine disease other than diabetes; gangrene or active sepsis; severe retinopathy; nondiabetic renal disease or renal allograft; malignancy, except cutaneous basal cell carcinoma; or known abnormality of Cu or Fe metabolism as well as current treatment with diuretics or calcium-channel blockers.
All protocols received appropriate regulatory approval, and all subjects provided written informed consent.
Elemental balance studies.
Type 2 diabetic (n = 20) and control (n = 20) subjects underwent a factorial, randomized, double-blind, placebo-controlled elemental balance study with screening, enrollment, run-in, treatment, and follow-up periods. Participants were resident throughout the study. Elemental intake was controlled by providing all items of food and beverages, which were directly measured (duplicate diets). Diets (Xyris Foodworks) were constructed to adhere to American Diabetes Association recommendations (9).
Elemental excretion was determined throughout the basal period. Balances were calculated from the difference between intake and output. [Elements]fasting serum were determined on the mornings of days 1 and 7, the latter before drugs were administered. After the basal study (days 1eC6), subjects were randomized to placebo or trientine (2,400 mg/day; Anstead, Essex, U.K.) and immediately entered the 6-day treatment period (days 7eC12). The second part of the study was an otherwise identical regimen, which was completed on the morning of day 13.
Sample acquisition and analysis.
Completeness of urine collections was confirmed by a p-aminobenzoic acid (PABA) test. Feces were freeze-dried; completeness was considered to be 93eC97% (opaque food-markers; X-ray of fecal samples). Elemental concentrations in urine, feces, and food aliquots were measured (10) using an inductively coupled plasmaeCmass spectrometer (Perkin-Elmer Sciex Elan 6100). Biochemical and hematological variables (Table 1) were measured in fasting blood samples on days 1 and 7 (pretreatment) and day 13 (treatment). The level of 2-h [Mg]serum was determined for 10-h after dosing. Data reported for day 1 are herein termed "basal" and those for days 1 and 7 are designated "pretreatment."
Dosage-dependent urinary metal excretion.
We also measured the effects of increasing dosages of trientine (300, 600, 1,200, and 2,400 mg/day) on urinary elements. Samples were collected before and after each 1-week treatment period from control and diabetic subjects (n = 7 each) after completion of the main study (total duration 28 weeks).
Statistical analysis.
Basal values from healthy (control) and diabetic patients were compared by one-way ANOVA. Restrictive iterative generalized least squares (RIGLS) models were fitted by restricted maximum likelihood (REML) (11). The effects of patient status (control or diabetic) and treatment (placebo or trientine) were analyzed using a factorial experimental design with two time periods, days 0eC6 and 7eC12, wherein the interaction effect was the term of primary significance. Differences between time periods for each subject were used in a mixed-model ANOVA fitted by REML in which subjects were considered as random (12) and differences between treatments (trientine or placebo) and patient status (diabetic or control) and the interaction between treatment and patient status were tested. Assumptions of normality and homoscedasticity were directly verified. Pearson’s correlation coefficients were calculated to determine the relation between basal Cu and key variables. A model predicting urinary Cu excretion during drug treatment (days 7eC12) was constructed by adding predictive basal (day 1) variables into a forward, stepwise, multiple regression model (with = 0.25 for entry and = 0.10 for leaving). A significance level of = 0.05 was used for all statistical tests.
RESULTS
Basal group characteristics.
In all, 20 diabetic and 19 control subjects completed the study. Diabetic subjects had greater BMI, fasting plasma glucose (FPG), and HbA1c than did control subjects (each P < 0.001) (Table 1). Basal [Mg]serum was lower (P < 0.01) and [ferritin]serum was elevated (P < 0.001) in diabetic compared with control subjects. Serum levels are usually not a good measure of nutritional status, and measured values of Cu and five other elements did not differ between diabetic and control subjects (Table 1).
Relation between serum HbA1c and the interaction between extracellular superoxide dismutase and [Cu]serum.
Basal extracellular superoxide dismutase (EC-SOD) was elevated in diabetic subjects on days 1 and 7 (both P < 0.01) and was related to [Cu]serum levels (EC-SOD = 19.6.[Cu]serum eC 228; r2 = 0.16, P = 0.011), but no equivalent association was observed in control subjects. Basal EC-SOD did not correlate with the serum concentration of any other element in diabetic subjects. EC-SOD was related to the interaction between [Cu]serum and HbA1c in RIGLS models on days 1 ([Cu]serum, P = 0.022; HbA1c, P = 0.014; interaction term, P = 0.0096) and 7 ([Cu]serum, HbA1c, and interaction term, all P < 0.0001). A three-dimensional spline-surface (on day 7) illustrated that maximum elevations in EC-SOD occurred mainly in subjects in whom both [Cu]serum and HbA1c were high (Fig. 1). Thus, elevated EC-SOD may reflect a chemical interaction between these variables. In diabetic subjects, trientine treatment lowered mean serum EC-SOD below baseline levels (31.8 ± 34.7 [day 13] vs. 72.6 ± 72.0 [day 1] units/l; P = 0.034). EC-SOD was not related to interactions between serum concentrations of any other elements and HbA1c in diabetic subjects (RIGLS; all NS) nor was HbA1c alone significantly related to EC-SOD in either diabetic or control subjects.
Serum ferritin.
[Ferritin]serum was elevated in diabetic compared with control subjects (Table 1), but values were uncorrelated with [Fe]serum, iron-binding capacity (IBC), [Hb]blood, or packed cell volume in diabetic subjects. These findings are consistent with those in previous reports.
Basal elemental balance.
Basal elemental balances are presented in Table 2. Food intake did not differ between groups or as a result of treatment, nor did the basal balance differ between control and diabetic groups. The Cu balance was variable but tended to be more positive in diabetic than in control subjects, although the difference was not significant (P = 0.19) (Table 2). Basal urinary excretion of Cu, Fe, Zn, Ca, Mn, Se, and Cr was higher in diabetic subjects, and basal urinary Cu excretion was closely correlated with that of Fe ([Fe]24 h urine = 1.94.[Cu]24 h urine + 0.53; r2 = 0.48, P = 0.00067). Thus, increased basal urinary Cu excretion in diabetes is closely related to increased urinary Fe excretion. Diabetic subjects had elevated urinary volumes, but statistical modeling indicated that the increased urinary output was not a factor in the increased urinary elemental outputs.
Effects of drug treatment on elemental balance.
The effects of drug treatment (placebo or trientine) and of interactions between trientine and metabolic status (control or diabetes) were examined by ANOVA (Table 3); Table 4 shows the effects of treatment on differences in balance and urinary and fecal excretion of Cu, Fe, Zn, and Ca.
Copper.
Trientine did not modify the Cu balance in the whole study group, but the interaction term was significant (P = 0.0028) (Table 3). Trientine decreased the Cu balance in diabetic subjects compared with placebo (P = 0.021) (Table 4), but its effect on balance in control subjects was of marginal significance (P = 0.065) (Table 4). Basal urinary Cu was elevated in diabetic subjects (P = 0.0011) (Table 2). Trientine treatment modified urinary Cu excretion in all subjects (P < 0.0001), but the interaction was borderline (P = 0.075) (Table 3). Trientine stimulated urinary Cu excretion in both groups (P < 0.0001) (Table 4), indicating extraction of CuII in both. Moreover, its interaction with fecal Cu was significant (P = 0.0034) (Table 3). Fecal Cu effects were evoked largely via decreased control values (P = 0.0041) (Table 4). These observations are consistent with lower fractional absorption of Cu in diabetes.
Iron.
Trientine modified the Fe balance (P = 0.028), but the corresponding trientineeCmetabolic status interaction term was not significant (Table 3). Trientine increased the Fe balance in control (P = 0.034) but not in diabetic subjects (Table 4). Basal urinary Fe was elevated in diabetic subjects (P = 0.0001) (Table 2), but was unaffected by trientine, which had only borderline effects in diabetic subjects (P = 0.051) (Table 4). Trientine modified fecal Fe in the whole group (P = 0.019) (Table 3), but the trientineeCmetabolic status interaction was insignificant. Trientine lowered fecal Fe excretion in control subjects (P = 0.023) (Table 4). These data suggest lower fractional absorption of Fe in diabetes.
Zinc.
Trientine elicited significant modification of the Zn balance in all subjects (P = 0.0021), and the trientine-metabolic status interaction was significant (P = 0.002) (Table 3). Basal urinary Zn was elevated in diabetic subjects (P < 0.0001) (Table 2). Trientine modified urinary Zn in all subjects (P < 0.0001) (Table 3) by stimulation in both control (P < 0.0001) and diabetic (P < 0.0001) subjects (Table 4). Trientine modified fecal Zn excretion (P < 0.0001) (Table 3), and the trientineeCmetabolic status interaction was significant (P = 0.0045). Trientine decreased fecal Zn in control subjects (P = 0.0001) (Table 4). These data suggest that fractional Zn absorption is lower in diabetes.
Calcium.
Trientine significantly modified the Ca balance (P = 0.0022) (Table 3), mainly by increases in control subjects (P = 0.0012) (Table 4). DrugeCmetabolic status interactions were significant for Ca balance (P = 0.033) and fecal excretion (P = 0.020) (Table 3). Trientine modified fecal Ca excretion (P = 0.0014) (Table 3), mainly via decreased control values (P = 0.00065) (Table 4). Fractional Ca absorption may thus be lower in diabetes.
Magnesium.
The basal Mg balance did not differ between diabetic and control subjects, nor was it modified by trientine treatment. Indexes of Mg balance were uncorrelated with HbA1c or FPG in either group (data not shown).
Manganese.
Trientine modified the Mn balance (P = 0.028) and fecal Mn excretion (P = 0.017) (Table 3). The trientineeCmetabolic status interaction was significant for Mn balance (P = 0.043) and fecal excretion (P = 0.034) (Table 3). Trientine altered the Mn balance mainly via decreased fecal excretion. The effects on Mn and Ca balance were similar, being evoked mainly via decreased fecal excretion in control subjects and consistent with decreased fractional gut absorption of Mn in diabetic subjects.
Selenium.
The trientineeCmetabolic status interaction was significant for Se balance (P = 0.016) and fecal excretion (P = 0.013) (Table 3). The effects on Se balance resembled those for Ca and Mn, being evoked mainly via decreased fecal Se excretion in control subjects. Trientine did not modify [Se]serum (data not shown).
Molybdenum/chromium.
Neither diabetes nor trientine altered the Mo or Cr balance or their urinary or fecal excretion rates (data not shown). Neither FPG nor HbA1c correlated with indexes of Cr balance in either diabetic or control subjects (data not shown).
Dosage-dependent effects of trientine on metal excretion.
Trientine increased urinary Cu in both diabetic ([Cu]24 h urine = 0.00245.[trientine dosage] + 0.192; r2 = 0.66, P < 0.0001) and nondiabetic ([Cu]24 h urine = 0.00183.[trientine dosage] + 0.177; r2 = 0.56, P < 0.0001) subjects in a dosage-dependent manner; gradients for Cu excretion did not differ between groups. Trientine stimulated urinary Zn in both diabetic ([Zn]24 h urine = 0.016.[trientine dosage] + 14.5; r2 = 0.54, P < 0.0001) and nondiabetic ([Zn]24 h urine = 0.0097.[trientine dosage] + 4.32; r2 = 0.42, P < 0.0001) groups in a dosage-dependent manner; the diabetic gradient was significantly greater (P < 0.05). These findings are consistent with those previously reported (13). Trientine had no dosage-dependent effect on 24-h urinary excretion of Fe, Mn, Ca, Mg, Mo, Se, or Cr (data not shown), nor did it modify [Mg]serum over 10 h (data not shown).
Basal variables predicting cupriuretic response to trientine.
We calculated a multivariate regression model relating basal variables to urinary Cu excretion during drug treatment (Table 5). Trientine and diabetes were positive factors; urinary Cu excretion on days 1eC6 and basal [Mg]serum were significant, positively continuous components and basal [ferritin]serum was negative. The mechanisms relating basal [Mg]serum and [ferritin]serum to urinary Cu are uncertain.
DISCUSSION
In this study, we found that several aspects of Cu metabolism were altered in diabetic subjects. Basal urinary Cu was 1.4-fold higher in diabetic than in control subjects, whereas [Cu]serum did not differ significantly between the two groups. These findings are consistent with some previous reports (14,15), but others have reported that diabetic subjects with complications have elevated [Cu]serum (16).
There was a trend for the Cu balance to be elevated in diabetic subjects, as well as a significant effect of interaction between trientine and diabetes on Cu balance. Trientine markedly stimulated urinary Cu in both subject groups, but lowered the Cu balance only in diabetic subjects. Urinary Cu excretion during drug treatment was positively correlated with pretreatment urinary Cu levels. Thus, elevated basal urinary Cu predicted drug-induced cupriuresis; individual responses may have been determined by prior systemic CuII accumulation. In contrast, trientine decreased fecal Cu in control subjects only, possibly through increased uptake, consistent with known actions of polyvalent chelators to increase metal absorption (17). An alternative mechanism that could contribute to the observed effects of trientine on fecal metal excretion is that diabetes may modify hepatobiliary excretion of some or all of the elements concerned, although we are unaware of data to support this hypothesis. Thus, regulation of Cu homeostasis differed significantly between groups, and trientine elicited effects to reverse the elevated Cu balance in diabetic subjects, mainly through stimulation of urinary Cu excretion.
Ceruloplasmin (ferro-O2-oxidoreductase; EC 1.16.3.1) is a circulating Cu protein present in vertebrate plasma (18). Normally, 95% of plasma Cu is ceruloplasmin bound, and levels of circulating Cu and ceruloplasmin are closely related (19). These are the biomarkers most frequently used as measures of Cu status, and both are depressed in severe Cu deficiency (20). However, levels plateau when Cu intake is adequate and do not reflect the magnitude of Cu intake beyond this point, so they are not useful for characterizing Cu excess states (21). In one study, neither biomarker decreased in humans fed a marginally low Cu diet for >3 months (22). Based on our study, we conclude that measurements of isolated [Cu]serum or [cerulo-plasmin]serum values are unlikely to be informative concerning the presence of probable systemic CuII excess in diabetic subjects.
Although Cu is an essential trace nutrient, it is also a potent cytotoxin when excess amounts accumulate in tissues (23). Because of its redox chemistry, CuII readily participates in reactions that elicit ROS production (4,24). Possible roles for Cu-catalyzed redox reactions in diabetes complications have been discussed previously (3,5), and the relative importance of Cu in diabetes in vivo was shown when we demonstrated the reversal of heart failure by systemic CuII chelation in diabetic rats (8).
In our study, EC-SOD activity was elevated in diabetic subjects and correlated with an HbA1c-[Cu]serum interaction. This finding is consistent with a mechanism whereby elevated EC-SOD is related to a [Cu]serumeCchronic hyperglycemia association, with the latter factor being accepted as a driver of the tendency to develop diabetes complications (25). EC-SOD, a secretory glycoprotein, is the major SOD isoenzyme in extracellular fluids (26) and blood vessel walls (27). Activity and concentrations of serum EC-SOD are known to be elevated in diabetic subjects (26,28), and a significant association between the serum concentration of EC-SOD and the severity of vascular complications has been previously reported (29). A correlation between [Cu]serum and EC-SOD activity has been reported in humans with essential hypertension (30). Serum EC-SOD activity is reportedly correlated with diabetes duration, carotid artery intimal-media thickness, and severity of nephropathy and retinopathy. It has also been proposed as a marker of vascular injury, possibly reflecting hyperglycemia-induced oxidative injury to the vascular endothelium (29). Nitric oxide (NO), a key physiological vasodilator (31), reacts with superoxide anion at an extremely rapid rate (1,27). The balance between superoxide anion concentrations and cellular antioxidant capacity, particularly SOD activity, is likely to regulate the bioactivity of NO (31). As the major SOD isoform present in vascular endothelium, where it acts to regulate superoxide levels, EC-SOD is a key regulator of endothelium-derived NO bioactivity in blood vessels (27).
Elevated EC-SOD levels (26,28) reflect increased superoxide production in diabetes (32). The finding of decreased EC-SOD after trientine treatment is consistent with the suppression of vascular superoxide production, and our current study implicates an interaction between [Cu]serum and chronic hyperglycemia in the mechanism by which diabetes causes vascular damage. Trientine treatment could suppress intravascular consumption of NO by lowering vascular superoxide production, thereby enhancing physiological vasodilatation, which is defective in diabetes (32). Trientine treatment lowered EC-SOD activity to control values in diabetic subjects. These data, taken in conjunction with data from our previous study demonstrating that chronic trientine treatment reverses established heart failure in diabetic rats and left ventricular hypertrophy in diabetic humans and rats (8), support the therapeutic lowering of systemic CuII for suppression of vascular damage. Because disease mechanisms in hypertensive heart disease, ischemic cardiomyopathy, and atherosclerosis are similar to those in diabetic heart disease (1,7), we expect that our results could also prove relevant in these other related conditions (8).
Basal balances of nine essential elements (33eC35) were equivalent between study groups, although the basal urinary excretion rates for Cu, Fe, Zn, Ca, Mn, Se, and Cr were significantly elevated in the diabetic group. Trientine treatment significantly increased the Zn balance in control subjects, mainly via decreased fecal excretion, whereas it stimulated urinary Zn output in both groups. Previous findings of hyperzincuria and lower Zn absorption in diabetic animals and humans have prompted conjecture that diabetic subjects might be more susceptible to Zn deficiency (36); however, others have reported increased tissue Zn values after trientine treatment (37). Apparent absorption and retention of Cu and Zn have been reported in diabetic rats in which food consumption was twice that of controls, and the fractional absorption of Zn and Cu was reported as being lower. In one study (37), net absorption was higher, although it was offset by higher urinary excretion so that the final Cu/Zn retention was similar in both groups. These findings are similar to our results. Low fractional absorption of Zn in diabetic rats has been attributed to lower intestinal transport associated with increased concentrations of intestinal metallothionein, an inhibitor of Cu and Zn transport (36). The increased metallothionein content in the enterocytes of diabetic patients is a plausible mechanism for the lesser degree of stimulation by trientine treatment of Cu and Zn uptake in diabetic subjects. This mechanism could protect against increased systemic uptake that might otherwise result from diabetic hyperphagia. We observed similar disparities in the effects of trientine on the uptake of Ca and Fe between control and diabetic subjects. Similar mechanisms may thus operate in diabetes to lessen the increased dietary Ca and Fe absorption that could otherwise result from hyperphagia. However, these effects are not likely to be mediated through increased enterocyte metallothionein, which appears not to bind to either Fe or Ca but operate through different, specific mechanisms.
Consistent with previous reports (38), mean serum ferritin was increased in diabetic subjects, although other measures of Fe homeostasis including serum Fe and IBC (39) were not different from control values. Basal urinary Fe excretion was also elevated in diabetic subjects, whereas fecal Fe and the Fe balance were similar. These observations could be explained by the increased metal uptake secondary to relative hyperphagia in the diabetic subjects before the imposition of strict dietary control during the controlled balance studies. Trientine’s effects on uptake of Ca, Mn, and Se were similar to those for Fe. At present, however, the significance of high plasma ferritin concentrations in a subset of diabetic patients remains unclear (40), as does the mechanism of the negative correlation between ferritin and [Cu]urine observed here in trientine-treated diabetic subjects. In the Third National Health and Nutrition Examination Survey (1988eC1994), an increased risk of diabetes was concentrated among participants with the lowest transferrin saturation concentrations; it was reported that this association was less likely to be explained by the Fe overload hypothesis and instead may be caused by inflammation as the mechanism by which ferritin becomes elevated in diabetes (38).
In our study we have shown that several alterations in the regulation of Cu homeostasis occur in diabetic humans. How might Cu and chronic hyperglycemia conspire to cause cardiovascular damage in diabetes Ceruloplasmin and serum albumin are the main Cu-binding proteins in plasma (41), and there is some evidence that chronic hyperglycemia can damage the Cu-binding properties of both (42,43). Results from our recent studies have indicated that diabetes might cause two- to threefold increments in extracellular matrix Cu (8). Furthermore, incubation of ceruloplasmin with glucose reportedly causes fragmentation and time-dependent release of its bound CuII, which then appears to participate in a Fenton-type reaction to produce hydroxyl radicals (42). These data were interpreted as consistent with the conjecture that ROS may form by the Maillard reaction and in turn generate hydroxyl radicals via a Cu-dependent Fenton-type reaction (42). In addition, glycated serum albumin reportedly becomes pro-oxidant in the presence of Cu, probably through the generation of ROS (44). Damage by chronic hyperglycemia to Cu-regulating mechanisms through glycation of ceruloplasmin and albumin could lead to elevated concentrations of catalytically active CuII in plasma, but this hypothesis requires support by direct observation. Cu binds to transferrin in addition to ceruloplasmin and albumin (45); this interaction could also be important in the association of Cu, EC-SOD, and HbA1c.
In summary, the regulation of Cu homeostasis was altered in the diabetic subjects in this study, who demonstrated elevated rates of urinary Cu excretion and a tendency to increased Cu balance compared with control subjects. Treatment with the CuII-selective chelator, trientine, lowered Cu balance in the diabetic subjects, in whom elevated EC-SOD was strongly correlated with the [Cu]plasma-HbA1c interaction, implicating this in diabetic tissue damage. Trientine suppressed elevations of EC-SOD in diabetic subjects. In conjunction with therapies aimed at decreasing hyperglycemia, the use of compounds that can remove systemic CuII is proposed as a therapy for suppressing the tendency to cardiovascular complications in diabetes.
ACKNOWLEDGMENTS
This work was supported by the Endocore Research Trust and Protemix.
We thank T.B. Mulvey, G. Allen, S. Thornber, P. Misur, G. Muir, G. Kerr, G. Robinson, and P. Baker for assistance; and M.S. Cameron-Cooper, K.R. Mansford, P.J. Scott, T. Brittain, C.A. Tse, A.R. Phillips, P.D. Boyd, K. Wichmann, and D.J. Crossman for their discussions with us. G.C. acknowledges program support from the Foundation of Research, Science and Technology, New Zealand and the Health Research Council of New Zealand.
FOOTNOTES
G.J.S.C. is a stock shareholder in, has served on an advisory panel or board of, and has received grant research support from Protemix. Y.-K.C. is employed by and has received grant/research support from Protemix. G.D.G. has received fees for providing statistical support to Protemix. J.R.B. is employed by, has served on the advisory panel or board of, and is a stock shareholder in Protemix. D.H.B. has been paid a consulting fee for statistical analysis by Protemix. S.D.P. has received grant/research support from Protemix. C.M.F. is associated with Protemix. Protemix is not affiliated with Anstead U.K., Ltd.
EC-SOD, extracellular superoxide dismutase; FPG, fasting plasma glucose; IBC, iron-binding capacity; PABA, p-aminobenzoic acid; REML, restricted maximum likelihood; RIGLS, restrictive iterative generalized least squares; ROS, reactive oxygen species
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2 Centre for Molecular Biodiscovery, Faculty of Science, University of Auckland, New Zealand
3 Department of Medicine, Faculty of Medical and Health Sciences, University of Auckland, New Zealand
4 Human Nutrition Unit, University of Auckland, New Zealand
5 South Auckland Diabetes Service, Middlemore Hospital, Otahuhu, New Zealand
6 School of Medical Sciences, Faculty of Medical and Health Sciences, University of Auckland, New Zealand
7 Department of Medicine, Christchurch Clinical School, University of Otago, Christchurch, New Zealand
ABSTRACT
We recently showed that treatment with the CuII-selective chelator, trientine, alleviates heart failure in diabetic rats, improves left ventricular hypertrophy in humans with type 2 diabetes, and increases urinary Cu excretion in both diabetic rats and humans compared with nondiabetic control subjects. In this study, we characterized the homeostasis of Cu and eight other nutritionally essential elements in diabetes under fully residential conditions in male subjects with type 2 diabetes and age-matched control subjects. We then probed elemental balance with oral trientine in a parallel-group, placebo-controlled study in these subjects. Before treatment, there were no detectable between-group differences in the balance of any element, although urinary output of several elements was greater in diabetic subjects. Mean extracellular superoxide dismutase (EC-SOD) activity was elevated in diabetic subjects, and its activity correlated strongly with the interaction between [Cu]serum and HbA1c. Trientine caused the Cu balance to become negative in diabetic subjects through elevated urinary Cu losses and suppressed elevated EC-SOD. Basal urinary Cu predicted urinary Cu losses during treatment, which caused extraction of systemic CuII. We suggest that cardiovascular complications in diabetes might be better controlled by therapeutic strategies that focus on lowering plasma glucose and loosely bound systemic CuII.
Oxidative stress has been implicated as a contributory mechanism in age-related disorders, including diabetes, hypertension, obesity, and atherosclerosis (1). Clinical trials of antioxidant (2) or carbonyl-trapping (3) agents in these disorders have had mixed success, indicating that the mechanisms underlying these disorders may be more complex than previously thought (3). In diabetes, the nature of oxidant stress and how it might promote complications is still unclear.
Free Fe and Cu ions are highly redox active (4) and might contribute to tissue damage by the generation of reactive oxygen species (ROS) (1), but the in vivo availability of catalytic Fe and Cu is usually very restricted (4). Both metals have been previously discussed in relation to the mechanisms of diabetes complications (5), but abnormalities of Fe homeostasis have not been linked to the major classes of diabetes and it is unknown whether altered Cu metabolism is relevant to diabetes complications.
Heart disease leads to death in most diabetic subjects (6,7). The Cu-selective chelator, trientine, causes a CuII-trientine complex to appear in the urine of diabetic rats whose abnormal Cu metabolism occurred after diabetes induction (8); trientine alleviated heart failure, improved cardiomyocyte structure, and reversed elevations in left ventricle collagen and 1 integrin without lowering blood glucose (8). Trientine also increased Cu excretion and decreased left ventricle mass in diabetic humans (8), implicating increased systemic CuII in the mechanism by which diabetes damages the heart.
Here we detail measurements of a 6-day balance of nine elements, including Cu and Fe, in diabetic and age-matched nondiabetic control subjects in whom we probed systemic metal balance with oral trientine in a subsequent study. Basal urinary output of Cu and Fe was significantly increased in diabetes, and those output values correlated strongly. Trientine treatment increased urinary excretion of Cu in a dosage-dependent manner, as predicted by basal urinary Cu, thereby causing a positive Cu balance to become negative in diabetes. In contrast, it modified neither Fe balance nor rates of urinary or fecal Fe excretion in these subjects. Furthermore, trientine did not render negative the balance of any other element in either diabetic or control subjects. Regulation of Cu metabolism was shown to be abnormal in diabetes and selectively modified by trientine, which did not concomitantly modify Fe metabolism. These findings are consistent with the effects of trientine in reversing the tendency to systemic accumulation of increased, loosely bound CuII in diabetes, which may help explain its therapeutic effects in diabetic cardiovascular disease.
RESEARCH DESIGN AND METHODS
Men (age 30eC70 years) with normal electrocardiograms were recruited for this study. Diabetic subjects had been diagnosed at least 6 months prior to the study, and control subjects had normal glucose tolerance. Exclusion criteria included the presence of type 1 diabetes; nephropathy; abnormal hematology or Fe deficiency; a history of significant cardiac disease; previous hepatic, gastrointestinal, or endocrine disease other than diabetes; gangrene or active sepsis; severe retinopathy; nondiabetic renal disease or renal allograft; malignancy, except cutaneous basal cell carcinoma; or known abnormality of Cu or Fe metabolism as well as current treatment with diuretics or calcium-channel blockers.
All protocols received appropriate regulatory approval, and all subjects provided written informed consent.
Elemental balance studies.
Type 2 diabetic (n = 20) and control (n = 20) subjects underwent a factorial, randomized, double-blind, placebo-controlled elemental balance study with screening, enrollment, run-in, treatment, and follow-up periods. Participants were resident throughout the study. Elemental intake was controlled by providing all items of food and beverages, which were directly measured (duplicate diets). Diets (Xyris Foodworks) were constructed to adhere to American Diabetes Association recommendations (9).
Elemental excretion was determined throughout the basal period. Balances were calculated from the difference between intake and output. [Elements]fasting serum were determined on the mornings of days 1 and 7, the latter before drugs were administered. After the basal study (days 1eC6), subjects were randomized to placebo or trientine (2,400 mg/day; Anstead, Essex, U.K.) and immediately entered the 6-day treatment period (days 7eC12). The second part of the study was an otherwise identical regimen, which was completed on the morning of day 13.
Sample acquisition and analysis.
Completeness of urine collections was confirmed by a p-aminobenzoic acid (PABA) test. Feces were freeze-dried; completeness was considered to be 93eC97% (opaque food-markers; X-ray of fecal samples). Elemental concentrations in urine, feces, and food aliquots were measured (10) using an inductively coupled plasmaeCmass spectrometer (Perkin-Elmer Sciex Elan 6100). Biochemical and hematological variables (Table 1) were measured in fasting blood samples on days 1 and 7 (pretreatment) and day 13 (treatment). The level of 2-h [Mg]serum was determined for 10-h after dosing. Data reported for day 1 are herein termed "basal" and those for days 1 and 7 are designated "pretreatment."
Dosage-dependent urinary metal excretion.
We also measured the effects of increasing dosages of trientine (300, 600, 1,200, and 2,400 mg/day) on urinary elements. Samples were collected before and after each 1-week treatment period from control and diabetic subjects (n = 7 each) after completion of the main study (total duration 28 weeks).
Statistical analysis.
Basal values from healthy (control) and diabetic patients were compared by one-way ANOVA. Restrictive iterative generalized least squares (RIGLS) models were fitted by restricted maximum likelihood (REML) (11). The effects of patient status (control or diabetic) and treatment (placebo or trientine) were analyzed using a factorial experimental design with two time periods, days 0eC6 and 7eC12, wherein the interaction effect was the term of primary significance. Differences between time periods for each subject were used in a mixed-model ANOVA fitted by REML in which subjects were considered as random (12) and differences between treatments (trientine or placebo) and patient status (diabetic or control) and the interaction between treatment and patient status were tested. Assumptions of normality and homoscedasticity were directly verified. Pearson’s correlation coefficients were calculated to determine the relation between basal Cu and key variables. A model predicting urinary Cu excretion during drug treatment (days 7eC12) was constructed by adding predictive basal (day 1) variables into a forward, stepwise, multiple regression model (with = 0.25 for entry and = 0.10 for leaving). A significance level of = 0.05 was used for all statistical tests.
RESULTS
Basal group characteristics.
In all, 20 diabetic and 19 control subjects completed the study. Diabetic subjects had greater BMI, fasting plasma glucose (FPG), and HbA1c than did control subjects (each P < 0.001) (Table 1). Basal [Mg]serum was lower (P < 0.01) and [ferritin]serum was elevated (P < 0.001) in diabetic compared with control subjects. Serum levels are usually not a good measure of nutritional status, and measured values of Cu and five other elements did not differ between diabetic and control subjects (Table 1).
Relation between serum HbA1c and the interaction between extracellular superoxide dismutase and [Cu]serum.
Basal extracellular superoxide dismutase (EC-SOD) was elevated in diabetic subjects on days 1 and 7 (both P < 0.01) and was related to [Cu]serum levels (EC-SOD = 19.6.[Cu]serum eC 228; r2 = 0.16, P = 0.011), but no equivalent association was observed in control subjects. Basal EC-SOD did not correlate with the serum concentration of any other element in diabetic subjects. EC-SOD was related to the interaction between [Cu]serum and HbA1c in RIGLS models on days 1 ([Cu]serum, P = 0.022; HbA1c, P = 0.014; interaction term, P = 0.0096) and 7 ([Cu]serum, HbA1c, and interaction term, all P < 0.0001). A three-dimensional spline-surface (on day 7) illustrated that maximum elevations in EC-SOD occurred mainly in subjects in whom both [Cu]serum and HbA1c were high (Fig. 1). Thus, elevated EC-SOD may reflect a chemical interaction between these variables. In diabetic subjects, trientine treatment lowered mean serum EC-SOD below baseline levels (31.8 ± 34.7 [day 13] vs. 72.6 ± 72.0 [day 1] units/l; P = 0.034). EC-SOD was not related to interactions between serum concentrations of any other elements and HbA1c in diabetic subjects (RIGLS; all NS) nor was HbA1c alone significantly related to EC-SOD in either diabetic or control subjects.
Serum ferritin.
[Ferritin]serum was elevated in diabetic compared with control subjects (Table 1), but values were uncorrelated with [Fe]serum, iron-binding capacity (IBC), [Hb]blood, or packed cell volume in diabetic subjects. These findings are consistent with those in previous reports.
Basal elemental balance.
Basal elemental balances are presented in Table 2. Food intake did not differ between groups or as a result of treatment, nor did the basal balance differ between control and diabetic groups. The Cu balance was variable but tended to be more positive in diabetic than in control subjects, although the difference was not significant (P = 0.19) (Table 2). Basal urinary excretion of Cu, Fe, Zn, Ca, Mn, Se, and Cr was higher in diabetic subjects, and basal urinary Cu excretion was closely correlated with that of Fe ([Fe]24 h urine = 1.94.[Cu]24 h urine + 0.53; r2 = 0.48, P = 0.00067). Thus, increased basal urinary Cu excretion in diabetes is closely related to increased urinary Fe excretion. Diabetic subjects had elevated urinary volumes, but statistical modeling indicated that the increased urinary output was not a factor in the increased urinary elemental outputs.
Effects of drug treatment on elemental balance.
The effects of drug treatment (placebo or trientine) and of interactions between trientine and metabolic status (control or diabetes) were examined by ANOVA (Table 3); Table 4 shows the effects of treatment on differences in balance and urinary and fecal excretion of Cu, Fe, Zn, and Ca.
Copper.
Trientine did not modify the Cu balance in the whole study group, but the interaction term was significant (P = 0.0028) (Table 3). Trientine decreased the Cu balance in diabetic subjects compared with placebo (P = 0.021) (Table 4), but its effect on balance in control subjects was of marginal significance (P = 0.065) (Table 4). Basal urinary Cu was elevated in diabetic subjects (P = 0.0011) (Table 2). Trientine treatment modified urinary Cu excretion in all subjects (P < 0.0001), but the interaction was borderline (P = 0.075) (Table 3). Trientine stimulated urinary Cu excretion in both groups (P < 0.0001) (Table 4), indicating extraction of CuII in both. Moreover, its interaction with fecal Cu was significant (P = 0.0034) (Table 3). Fecal Cu effects were evoked largely via decreased control values (P = 0.0041) (Table 4). These observations are consistent with lower fractional absorption of Cu in diabetes.
Iron.
Trientine modified the Fe balance (P = 0.028), but the corresponding trientineeCmetabolic status interaction term was not significant (Table 3). Trientine increased the Fe balance in control (P = 0.034) but not in diabetic subjects (Table 4). Basal urinary Fe was elevated in diabetic subjects (P = 0.0001) (Table 2), but was unaffected by trientine, which had only borderline effects in diabetic subjects (P = 0.051) (Table 4). Trientine modified fecal Fe in the whole group (P = 0.019) (Table 3), but the trientineeCmetabolic status interaction was insignificant. Trientine lowered fecal Fe excretion in control subjects (P = 0.023) (Table 4). These data suggest lower fractional absorption of Fe in diabetes.
Zinc.
Trientine elicited significant modification of the Zn balance in all subjects (P = 0.0021), and the trientine-metabolic status interaction was significant (P = 0.002) (Table 3). Basal urinary Zn was elevated in diabetic subjects (P < 0.0001) (Table 2). Trientine modified urinary Zn in all subjects (P < 0.0001) (Table 3) by stimulation in both control (P < 0.0001) and diabetic (P < 0.0001) subjects (Table 4). Trientine modified fecal Zn excretion (P < 0.0001) (Table 3), and the trientineeCmetabolic status interaction was significant (P = 0.0045). Trientine decreased fecal Zn in control subjects (P = 0.0001) (Table 4). These data suggest that fractional Zn absorption is lower in diabetes.
Calcium.
Trientine significantly modified the Ca balance (P = 0.0022) (Table 3), mainly by increases in control subjects (P = 0.0012) (Table 4). DrugeCmetabolic status interactions were significant for Ca balance (P = 0.033) and fecal excretion (P = 0.020) (Table 3). Trientine modified fecal Ca excretion (P = 0.0014) (Table 3), mainly via decreased control values (P = 0.00065) (Table 4). Fractional Ca absorption may thus be lower in diabetes.
Magnesium.
The basal Mg balance did not differ between diabetic and control subjects, nor was it modified by trientine treatment. Indexes of Mg balance were uncorrelated with HbA1c or FPG in either group (data not shown).
Manganese.
Trientine modified the Mn balance (P = 0.028) and fecal Mn excretion (P = 0.017) (Table 3). The trientineeCmetabolic status interaction was significant for Mn balance (P = 0.043) and fecal excretion (P = 0.034) (Table 3). Trientine altered the Mn balance mainly via decreased fecal excretion. The effects on Mn and Ca balance were similar, being evoked mainly via decreased fecal excretion in control subjects and consistent with decreased fractional gut absorption of Mn in diabetic subjects.
Selenium.
The trientineeCmetabolic status interaction was significant for Se balance (P = 0.016) and fecal excretion (P = 0.013) (Table 3). The effects on Se balance resembled those for Ca and Mn, being evoked mainly via decreased fecal Se excretion in control subjects. Trientine did not modify [Se]serum (data not shown).
Molybdenum/chromium.
Neither diabetes nor trientine altered the Mo or Cr balance or their urinary or fecal excretion rates (data not shown). Neither FPG nor HbA1c correlated with indexes of Cr balance in either diabetic or control subjects (data not shown).
Dosage-dependent effects of trientine on metal excretion.
Trientine increased urinary Cu in both diabetic ([Cu]24 h urine = 0.00245.[trientine dosage] + 0.192; r2 = 0.66, P < 0.0001) and nondiabetic ([Cu]24 h urine = 0.00183.[trientine dosage] + 0.177; r2 = 0.56, P < 0.0001) subjects in a dosage-dependent manner; gradients for Cu excretion did not differ between groups. Trientine stimulated urinary Zn in both diabetic ([Zn]24 h urine = 0.016.[trientine dosage] + 14.5; r2 = 0.54, P < 0.0001) and nondiabetic ([Zn]24 h urine = 0.0097.[trientine dosage] + 4.32; r2 = 0.42, P < 0.0001) groups in a dosage-dependent manner; the diabetic gradient was significantly greater (P < 0.05). These findings are consistent with those previously reported (13). Trientine had no dosage-dependent effect on 24-h urinary excretion of Fe, Mn, Ca, Mg, Mo, Se, or Cr (data not shown), nor did it modify [Mg]serum over 10 h (data not shown).
Basal variables predicting cupriuretic response to trientine.
We calculated a multivariate regression model relating basal variables to urinary Cu excretion during drug treatment (Table 5). Trientine and diabetes were positive factors; urinary Cu excretion on days 1eC6 and basal [Mg]serum were significant, positively continuous components and basal [ferritin]serum was negative. The mechanisms relating basal [Mg]serum and [ferritin]serum to urinary Cu are uncertain.
DISCUSSION
In this study, we found that several aspects of Cu metabolism were altered in diabetic subjects. Basal urinary Cu was 1.4-fold higher in diabetic than in control subjects, whereas [Cu]serum did not differ significantly between the two groups. These findings are consistent with some previous reports (14,15), but others have reported that diabetic subjects with complications have elevated [Cu]serum (16).
There was a trend for the Cu balance to be elevated in diabetic subjects, as well as a significant effect of interaction between trientine and diabetes on Cu balance. Trientine markedly stimulated urinary Cu in both subject groups, but lowered the Cu balance only in diabetic subjects. Urinary Cu excretion during drug treatment was positively correlated with pretreatment urinary Cu levels. Thus, elevated basal urinary Cu predicted drug-induced cupriuresis; individual responses may have been determined by prior systemic CuII accumulation. In contrast, trientine decreased fecal Cu in control subjects only, possibly through increased uptake, consistent with known actions of polyvalent chelators to increase metal absorption (17). An alternative mechanism that could contribute to the observed effects of trientine on fecal metal excretion is that diabetes may modify hepatobiliary excretion of some or all of the elements concerned, although we are unaware of data to support this hypothesis. Thus, regulation of Cu homeostasis differed significantly between groups, and trientine elicited effects to reverse the elevated Cu balance in diabetic subjects, mainly through stimulation of urinary Cu excretion.
Ceruloplasmin (ferro-O2-oxidoreductase; EC 1.16.3.1) is a circulating Cu protein present in vertebrate plasma (18). Normally, 95% of plasma Cu is ceruloplasmin bound, and levels of circulating Cu and ceruloplasmin are closely related (19). These are the biomarkers most frequently used as measures of Cu status, and both are depressed in severe Cu deficiency (20). However, levels plateau when Cu intake is adequate and do not reflect the magnitude of Cu intake beyond this point, so they are not useful for characterizing Cu excess states (21). In one study, neither biomarker decreased in humans fed a marginally low Cu diet for >3 months (22). Based on our study, we conclude that measurements of isolated [Cu]serum or [cerulo-plasmin]serum values are unlikely to be informative concerning the presence of probable systemic CuII excess in diabetic subjects.
Although Cu is an essential trace nutrient, it is also a potent cytotoxin when excess amounts accumulate in tissues (23). Because of its redox chemistry, CuII readily participates in reactions that elicit ROS production (4,24). Possible roles for Cu-catalyzed redox reactions in diabetes complications have been discussed previously (3,5), and the relative importance of Cu in diabetes in vivo was shown when we demonstrated the reversal of heart failure by systemic CuII chelation in diabetic rats (8).
In our study, EC-SOD activity was elevated in diabetic subjects and correlated with an HbA1c-[Cu]serum interaction. This finding is consistent with a mechanism whereby elevated EC-SOD is related to a [Cu]serumeCchronic hyperglycemia association, with the latter factor being accepted as a driver of the tendency to develop diabetes complications (25). EC-SOD, a secretory glycoprotein, is the major SOD isoenzyme in extracellular fluids (26) and blood vessel walls (27). Activity and concentrations of serum EC-SOD are known to be elevated in diabetic subjects (26,28), and a significant association between the serum concentration of EC-SOD and the severity of vascular complications has been previously reported (29). A correlation between [Cu]serum and EC-SOD activity has been reported in humans with essential hypertension (30). Serum EC-SOD activity is reportedly correlated with diabetes duration, carotid artery intimal-media thickness, and severity of nephropathy and retinopathy. It has also been proposed as a marker of vascular injury, possibly reflecting hyperglycemia-induced oxidative injury to the vascular endothelium (29). Nitric oxide (NO), a key physiological vasodilator (31), reacts with superoxide anion at an extremely rapid rate (1,27). The balance between superoxide anion concentrations and cellular antioxidant capacity, particularly SOD activity, is likely to regulate the bioactivity of NO (31). As the major SOD isoform present in vascular endothelium, where it acts to regulate superoxide levels, EC-SOD is a key regulator of endothelium-derived NO bioactivity in blood vessels (27).
Elevated EC-SOD levels (26,28) reflect increased superoxide production in diabetes (32). The finding of decreased EC-SOD after trientine treatment is consistent with the suppression of vascular superoxide production, and our current study implicates an interaction between [Cu]serum and chronic hyperglycemia in the mechanism by which diabetes causes vascular damage. Trientine treatment could suppress intravascular consumption of NO by lowering vascular superoxide production, thereby enhancing physiological vasodilatation, which is defective in diabetes (32). Trientine treatment lowered EC-SOD activity to control values in diabetic subjects. These data, taken in conjunction with data from our previous study demonstrating that chronic trientine treatment reverses established heart failure in diabetic rats and left ventricular hypertrophy in diabetic humans and rats (8), support the therapeutic lowering of systemic CuII for suppression of vascular damage. Because disease mechanisms in hypertensive heart disease, ischemic cardiomyopathy, and atherosclerosis are similar to those in diabetic heart disease (1,7), we expect that our results could also prove relevant in these other related conditions (8).
Basal balances of nine essential elements (33eC35) were equivalent between study groups, although the basal urinary excretion rates for Cu, Fe, Zn, Ca, Mn, Se, and Cr were significantly elevated in the diabetic group. Trientine treatment significantly increased the Zn balance in control subjects, mainly via decreased fecal excretion, whereas it stimulated urinary Zn output in both groups. Previous findings of hyperzincuria and lower Zn absorption in diabetic animals and humans have prompted conjecture that diabetic subjects might be more susceptible to Zn deficiency (36); however, others have reported increased tissue Zn values after trientine treatment (37). Apparent absorption and retention of Cu and Zn have been reported in diabetic rats in which food consumption was twice that of controls, and the fractional absorption of Zn and Cu was reported as being lower. In one study (37), net absorption was higher, although it was offset by higher urinary excretion so that the final Cu/Zn retention was similar in both groups. These findings are similar to our results. Low fractional absorption of Zn in diabetic rats has been attributed to lower intestinal transport associated with increased concentrations of intestinal metallothionein, an inhibitor of Cu and Zn transport (36). The increased metallothionein content in the enterocytes of diabetic patients is a plausible mechanism for the lesser degree of stimulation by trientine treatment of Cu and Zn uptake in diabetic subjects. This mechanism could protect against increased systemic uptake that might otherwise result from diabetic hyperphagia. We observed similar disparities in the effects of trientine on the uptake of Ca and Fe between control and diabetic subjects. Similar mechanisms may thus operate in diabetes to lessen the increased dietary Ca and Fe absorption that could otherwise result from hyperphagia. However, these effects are not likely to be mediated through increased enterocyte metallothionein, which appears not to bind to either Fe or Ca but operate through different, specific mechanisms.
Consistent with previous reports (38), mean serum ferritin was increased in diabetic subjects, although other measures of Fe homeostasis including serum Fe and IBC (39) were not different from control values. Basal urinary Fe excretion was also elevated in diabetic subjects, whereas fecal Fe and the Fe balance were similar. These observations could be explained by the increased metal uptake secondary to relative hyperphagia in the diabetic subjects before the imposition of strict dietary control during the controlled balance studies. Trientine’s effects on uptake of Ca, Mn, and Se were similar to those for Fe. At present, however, the significance of high plasma ferritin concentrations in a subset of diabetic patients remains unclear (40), as does the mechanism of the negative correlation between ferritin and [Cu]urine observed here in trientine-treated diabetic subjects. In the Third National Health and Nutrition Examination Survey (1988eC1994), an increased risk of diabetes was concentrated among participants with the lowest transferrin saturation concentrations; it was reported that this association was less likely to be explained by the Fe overload hypothesis and instead may be caused by inflammation as the mechanism by which ferritin becomes elevated in diabetes (38).
In our study we have shown that several alterations in the regulation of Cu homeostasis occur in diabetic humans. How might Cu and chronic hyperglycemia conspire to cause cardiovascular damage in diabetes Ceruloplasmin and serum albumin are the main Cu-binding proteins in plasma (41), and there is some evidence that chronic hyperglycemia can damage the Cu-binding properties of both (42,43). Results from our recent studies have indicated that diabetes might cause two- to threefold increments in extracellular matrix Cu (8). Furthermore, incubation of ceruloplasmin with glucose reportedly causes fragmentation and time-dependent release of its bound CuII, which then appears to participate in a Fenton-type reaction to produce hydroxyl radicals (42). These data were interpreted as consistent with the conjecture that ROS may form by the Maillard reaction and in turn generate hydroxyl radicals via a Cu-dependent Fenton-type reaction (42). In addition, glycated serum albumin reportedly becomes pro-oxidant in the presence of Cu, probably through the generation of ROS (44). Damage by chronic hyperglycemia to Cu-regulating mechanisms through glycation of ceruloplasmin and albumin could lead to elevated concentrations of catalytically active CuII in plasma, but this hypothesis requires support by direct observation. Cu binds to transferrin in addition to ceruloplasmin and albumin (45); this interaction could also be important in the association of Cu, EC-SOD, and HbA1c.
In summary, the regulation of Cu homeostasis was altered in the diabetic subjects in this study, who demonstrated elevated rates of urinary Cu excretion and a tendency to increased Cu balance compared with control subjects. Treatment with the CuII-selective chelator, trientine, lowered Cu balance in the diabetic subjects, in whom elevated EC-SOD was strongly correlated with the [Cu]plasma-HbA1c interaction, implicating this in diabetic tissue damage. Trientine suppressed elevations of EC-SOD in diabetic subjects. In conjunction with therapies aimed at decreasing hyperglycemia, the use of compounds that can remove systemic CuII is proposed as a therapy for suppressing the tendency to cardiovascular complications in diabetes.
ACKNOWLEDGMENTS
This work was supported by the Endocore Research Trust and Protemix.
We thank T.B. Mulvey, G. Allen, S. Thornber, P. Misur, G. Muir, G. Kerr, G. Robinson, and P. Baker for assistance; and M.S. Cameron-Cooper, K.R. Mansford, P.J. Scott, T. Brittain, C.A. Tse, A.R. Phillips, P.D. Boyd, K. Wichmann, and D.J. Crossman for their discussions with us. G.C. acknowledges program support from the Foundation of Research, Science and Technology, New Zealand and the Health Research Council of New Zealand.
FOOTNOTES
G.J.S.C. is a stock shareholder in, has served on an advisory panel or board of, and has received grant research support from Protemix. Y.-K.C. is employed by and has received grant/research support from Protemix. G.D.G. has received fees for providing statistical support to Protemix. J.R.B. is employed by, has served on the advisory panel or board of, and is a stock shareholder in Protemix. D.H.B. has been paid a consulting fee for statistical analysis by Protemix. S.D.P. has received grant/research support from Protemix. C.M.F. is associated with Protemix. Protemix is not affiliated with Anstead U.K., Ltd.
EC-SOD, extracellular superoxide dismutase; FPG, fasting plasma glucose; IBC, iron-binding capacity; PABA, p-aminobenzoic acid; REML, restricted maximum likelihood; RIGLS, restrictive iterative generalized least squares; ROS, reactive oxygen species
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