Zinc homeostasis in the metabolic syndrome and diabetes

Abstract

Zinc (Zn) is an essential mineral that is required for various cellular functions. Zn dyshomeostasis always is related to certain disorders such as metabolic syndrome, diabetes and diabetic complications. The associations of Zn with metabolic syndrome, diabetes and diabetic complications, thus, stem from the multiple roles of Zn: (1) a constructive component of many important enzymes or proteins, (2) a requirement for insulin storage and secretion, (3) a direct or indirect antioxidant action, and (4) an insulin-like action. However, whether there is a clear cause-and-effect relationship of Zn with metabolic syndrome, diabetes, or diabetic complications remains unclear. In fact, it is known that Zn deficiency is a common phenomenon in diabetic patients. Chronic low intake of Zn was associated with the increased risk of diabetes and diabetes also impairs Zn metabolism. Theoretically Zn supplementation should prevent the metabolic syndrome, diabetes, and diabetic complications; however, limited available data are not always supportive of the above notion. Therefore, this review has tried to summarize these pieces of available information, possible mechanisms by which Zn prevents the metabolic syndrome, diabetes, and diabetic complications. In the final part, what are the current issues for Zn supplementation were also discussed.

Keywords

zinc / zinc transporters / metallothionein / diabetes / diabetic complications / insulin resistance / antioxidant

Introduction

Zinc (Zn) is one of the important essential trace metals that are required for many cell events. Zn is not only an important nutrient, cofactor of numerous enzymes and transcription factors, but also acts as intracellular signaling mediator [1,2]. So far, more than 300 catalytically active Zn metalloproteins and more than 2000 Zn dependent transcription factors have been recognized; therefore, Zn is an integral component of a large variety of proteins and enzymes, and participates in a wide variety of metabolic processes including carbohydrate, lipid, protein and nucleic acid synthesis or degradation. Zn has diverse biological functions in enzymatic catalysis, redox regulation, cellular signal transduction, the immune system, and neurons. Therefore, Zn dyshomeostasis such as Zn deficiency is associated with various chronic pathogeneses.

Metabolic syndrome has been described for the first time by Reaven in 1988 [3], so that metabolic syndrome is also known as Reaven’s syndrome, metabolic syndrome X, cardiometabolic syndrome, syndrome X, insulin resistance syndrome (http://en.wikipedia.org/wiki/Metabolic_syndrome). Its prevalence increases regularly to reach more than 20% in the US adult population in the present time and increases with age [4]. Metabolic syndrome is a group of conditions that put people at risk for heart disease and diabetes. These conditions or called metabolic risk factors are (1)high blood pressure, (2)high blood sugar levels, (3)high levels of triglycerides, a type of fat, in the blood, (4)low levels of HDL, the good cholesterol, in the blood, (5)too much fat around the waist.

Diabetes mellitus (DM) is a major cause of morbidity and mortality world-wide, and also a major risk factor for early onset of coronary heart disease. In type 1 diabetes there is a lack of insulin production (i.e., insulin dependent DM, IDDM) and in type 2 diabetes the resistance to the effect of insulin is predominant (i.e., non-insulin dependent DM, NIDDM). Both type 1 and type 2 DM have long-term complications, including the detrimental effect of diabetes on the cardiovascular, renal, and nervous system, and the lower extremities, especially the feet [5]. Currently, treatment involves diet modification, weight reduction, exercise, oral medications, and insulin. Ongoing clinical trials testing of various medications to determine their effectiveness in treating the complications of diabetes have met with some success, but there still is much to learn about this disease. For instance, whether Zn supplementation can provide a therapeutic effect on diabetes and diabetic complications has been explored [6,7].

The present review, therefore, aims to summarize the information from our own studies and literatures, addressing the following issues [1]: the essentiality of Zn in respect with insulin [2]; Zn deficiency in metabolic syndrome and diabetes [3]; effect of Zn deficiency on cardiovascular diseases and other diabetic complications [4]; the effect of diabetes on Zn homeostasis [5]; effects of Zn supplementation on insulin resistance and diabetes [6]; effects of Zn supplementation on diabetic complications [7]; current issues that need to be solved and prospective.

The essentiality of Zn in respect with insulin

Zn, the second most prevalent trace element in the body, is involved in the structure and function of over 300 enzymes [2], collectively representing all major biochemical categories and thus is essential for normal cell function and metabolism. In our bodies the 85% of the whole body Zn are in muscle and bone, 11% in the skin and the liver, and the remaining in all the other tissue. In term of organs, the highest concentrations of Zn are in the prostate, retina, muscle, bone, liver, and kidney [2,8]. In multicellular organisms, virtually all Zn is intracellular. Among the intracellular Zn 30%-40% is located in the nucleus, 50% in the cytoplasm, organelles and specialized vesicles (for digestive enzymes or hormone storage) and the remainder in the cell membrane. Therefore, Zn in the tissue is not readily mobilized, i.e., no free Zn under normal conditions. Zn intake ranges from 107 to 231 μmol/d depending on the source, and the human Zn requirement is estimated at 15 mg/d. Zn has both catalytic and structural roles in enzymes, while in Zn finger motifs, it provides a scaffold that organizes protein sub-domains for the interaction with either DNA or other proteins. Zn plays a critical role for the function of a number of metalloproteins, inducing members of oxido-reductase, hydrolase, ligase, lyase family and has co-activating function with copper in superoxide dismutase or phospholipase C. The Zn ion (Zn²⁺) does not participate in redox reactions, which makes it a stable ion in a biological medium. Zn ions are hydrophilic and do not cross cell membranes by passive diffusion; therefore, Zn availability in the cells is dependent on its transporters [9]. In addition, metallothionein (MT), a cysteine-rich metal binding protein, also plays a critical role in Zn homeostasis [10-12]. Zn is excreted mostly in feces (12-15 mg/d) and lesser amounts (0.5 mg/d) in urine. Regarding the essentiality of Zn in details, several excellent reviews [2,13,14] can be found. In this section of the review, we will just summarize certain aspects of Zn related to metabolic syndrome, diabetes and diabetic complications.

The essentiality of Zn for insulin secretion

Pancreatic β-cells contain large amounts of Zn; one of the major roles of Zn is the binding of insulin in hexamers, a crystalline structure comprising two Zn ions and six insulin molecules, which are stored in the secretory granules [15]. Part of the Zn ion pool of the β-cell is co-secreted with insulin after stimulation with glucose. Immediately after secretion, the hexameric structure dissociates into the active monomer insulin and Zn ions, probably caused by a combination of a rapid decrease in Zn ion pressure and the change in pH from 5.5 to 7.4. An in vitro study suggested that the Zn ions co-secreted with insulin during hyperglycemia might contribute to β-cell death by a paracrine mechanism [16]; it has been hypothesized that such activity could link hyperinsulinism with β-cell necrosis and ensuing type 2 diabetes. The indispensable role of Zn for insulin structure makes it a requirement when insulin analog was prepared to ensure its activity and stabilization [16].

Zn transporters

To date, more than 20 Zn transporters were identified and characterized [17-19]. They are classified into two families: Zn transporter (ZnT: vertebrate cation diffusion facilitator family proteins, Slc30a family) and Zip (Zrt/Irt-like protein, Slc39a family). ZnT family members are responsible for the extrusion of Zn outside the cytoplasm to the extracellular space or intracellular compartments, while Zips move Zn in the opposite direction. The coordinated action of these two kinds of Zn transporters is essential to the maintenance of Zn homeostasis in the cytoplasm (Fig. 1), which plays important roles in the regulation of pathways [20].

A novel member of the ZnT family gene SLC30A8 was identified a decade ago [21]. ZnT8 is specifically expressed in the pancreatic β-cells and has been identified as a novel target autoantigen in patients with type 1 diabetes. Autoantibodies to ZnT8 (ZnT8A) are detected in 50%-60% of Japanese patients with acute-onset and 20% with slow-onset type 1 diabetes. In type 2 diabetes single nucleotide polymorphism in SLC30A8 (the gene of ZnT8), rs13266634 (Arg325Trp) has been reported in several studies [22-24]. These studies suggest the important role of ZnT, particularly ZnT8, in the development of diabetes.

MT

MTs are a group of intracellular cysteine-rich, low molecular weight (6-7 kDa) metal-binding proteins. Although four isoforms of MTs have been characterized, MT-I and MT-II are major isoforms found in most tissue. Intracellular Zn is strictly regulated by binding to MT by compartmentalization through the activities of ZnT. MTs have high binding affinity for Zn and play a central role in maintaining stable intracellular Zn availability through sequestration or release of Zn. These physical, chemical, and biological properties of MT have been summarized by several reviews [10,25,26].

Zn deficiency and metabolic syndrome and diabetes

Zn deficiency and insulin resistance and type 2 diabetes

Park et al. have used weanling male Sprague-Dawley rats to investigate the effect of Zn deficiency on glucose tolerance by intragastric force feeding to obviate decreased food intake and altered eating patterns. They demonstrated the induction of the glucose tolerance in rats fed a Zn-deficient diet. They further indicated that the glucose tolerance in rats on a Zn-deficient diet is not due to altered blood insulin and glucagon levels but rather to peripheral resistance to insulin action [27]. Later, other groups also showed the induction of insulin resistance in the animals fed with Zn-deficient diets [28,29].

The induction of insulin resistance by Zn deficiency was observed even in the offspring of maternal Zn deficiency. Rats consumed Zn-deficient (7 mg/g) or control (25 mg/g) diets ad libitum from 3-week preconception to 21-day postparturition. Litters were culled to 7 pups/dam postnatally and pups were allowed to be nursed by their original mothers; after weaning, pups were fed nonpurified diet. Insulin and glucose tolerance tests were performed on the pups at week 5 and 10. The authors found no difference in birth weight between groups; however, Zn-deficient pups weighed significantly more than controls by day 10 and 20. Both male and female Zn-deficient rats were less sensitive to insulin and glucose stimulation than controls at week 5 and 10. These findings suggest that suboptimal maternal Zn status induces long-term changes in the offspring related to abnormal glucose tolerance [29].

For human data, an early cross-sectional survey has been done to determine the association between current Zn intake with insulin resistance by recruiting 3575 subjects (25 to 64 years old), including 1769 rural (894 men, 875 women) and 1806 urban (904 men, 902 women) subjects. They found that 2-h plasma insulin levels also were associated with low Zn intake. Multivariate logistic regression analysis after adjustment for age showed that low level of Zn intake was inversely associated with central obesity, glucose intolerance, and diabetes in urban subjects. These associations were not observed in rural subjects [30]. Another study also suggests that Zn deficiency plays important roles in insulin resistance and subsequent hepatic fibrosis in patients with primary biliary cirrhosis, although insulin resistance in advanced stage primary biliary cirrhosis was significantly milder than that in HCV-related liver cirrhosis [31].

Low Zn associated with the development of diabetes

Since Zn plays a clear role in the synthesis, storage and secretion of insulin as well as conformational integrity of insulin in the hexameric form, Zn deficiency may be attributed to the pathogenesis of diabetes [32]. Epidemiological study has demonstrated that exposure to a low concentration of Zn in drinking water was associated with an increase in the risk for onset of IDDM. Using the Swedish childhood diabetes registry and data on residence 3 years before the onset of disease, a case-control study has shown that a high groundwater concentration of Zn was associated with a significant decrease in the risk, and low groundwater concentration of Zn was associated with a significant increase in the risk [33]. Later several other studies consistently supported this finding. For instance, Zhao et al. performed a similar study which covered the Cornwall and the former Plymouth Health Authority Regions in the far south-west of England, including 517 children with IDDM. They also found that the incidence rate of childhood diabetes is significantly associated with low Zn and magnesium in the drinking water [34]. Other recent studies further confirmed that low Zn in drinking water is associated with the risk of developing type 1 diabetes during childhood [35,36]. However, there was also one study that did not support the above findings. A Finland study showed that neither Zn nor nitrate and the urban/rural status of the area had a significant effect on the variation in incidence of IDDM during childhood [37]. The authors concluded that no significant effect might stem from the aggregated data being too crude to detect it, which suggests the complicated situation of epidemiology.

In studies using animal models the Zn deficiency that was induced with various Zn chelators was able to induce diabetes in some mammalian species, e.g., rabbits, mice, and hamsters, by β-cell destruction [38,39]. More interestingly, Zn deficiency increased the risk for diabetes in diabetes-prone experimental animals [40]. These studies with animal models clearly support the theory that Zn deficiency would be a risk factor for diabetic development.

Effect of Zn deficiency on cardiovascular diseases and other diabetic complications

To determine the association between Zn intake and prevalence of coronary artery disease, a cross-sectional survey was conducted and showed that the prevalence of coronary artery disease was significantly higher among subjects consuming lower intakes of dietary Zn. There was a higher prevalence of hypertension, hypertriglyceridemia and low high-density lipoprotein (HDL) cholesterol levels which showed significant upward trend with lower Zn intakes. Serum lipoprotein level also was associated with low Zn intake. Multivariate logistic regression analysis after adjustment for age showed that Zn intake and coronary artery disease were inversely associated. Serum Zn, serum triglycerides, blood pressure, and low HDL cholesterol were significant risk factors for coronary artery disease in urban subjects. These associations were not observed in rural subjects. They concluded that lower consumption of dietary Zn and low serum Zn levels were associated with an increased prevalence of coronary artery disease and several associated risk factors including hypertension and hypertriglyceridemia [30].

Lately Reiterer et al. have performed a very important study to test whether Zn deficiency can increase and Zn supplementation can decrease proatherosclerotic events in low-density lipoprotein (LDL) receptor knockout (LDL-R^{- / -} ) mice fed a moderate-fat diet [41]. They fed mice either a Zn-deficient (0 µmol Zn/g), a control (0.45 µmol Zn/g), or a Zn-supplemented (1.529 µmol Zn/g) diet for 4 weeks. Mice fed the Zn-deficient diet had significantly increased concentrations of cholesterol and triacylglycerides in the very low-density lipoprotein (VLDL) and HDL fractions. Zn supplementation decreased these lipid variables compared with control mice. A significantly higher concentration of glutathione reductase mRNA in the thoracic aortae was observed in Zn-deficient mice. Furthermore, inflammatory markers, such as nuclear factor-κB (NF-κB) and vascular cell adhesion molecule-1, were significantly increased in Zn-deficient mice compared with mice of the control or supplemented groups. In addition, Zn deficiency significantly reduced the DNA binding activity of peroxisome proliferator-activated receptors (PPARs) in liver extracts. Interestingly, mRNA expression levels of PPARγ were significantly increased in thoracic aortae of Zn-deficient mice, indicating an adaptation process to decreased PPAR signaling. These data provide important in vivo evidence of Zn deficiency inducing pro-inflammatory events in an atherogenic mouse model, suggesting that adequate Zn may be a critical component in protective PPAR signaling during atherosclerosis [41]. In a subsequent study they further obtained the following findings: (1) Zn deficiency increased plasma total cholesterol, which was also elevated by rosiglitazone (RSG); (2) Zn deficiency also caused an increased lipoprotein-cholesterol distribution toward the non-HDL fraction (VLDL, intermediate density lipoprotein, LDL); (3) Plasma total fatty acids tended to increase during Zn deficiency and RSG treatment resulted in similar changes in the fatty acid profile in Zn-deficient mice; (4) Fatty acid translocase (FAT/CD36) expression in abdominal aorta was upregulated by RSG only in Zn-deficient mice. In contrast, RSG treatment markedly increased lipoprotein lipase expression only in Zn-adequate mice. In addition, using an in vitro approach, they confirmed that adequate Zn is required for RSG-induced PPARγ activity to transcriptionally activate target genes. These data suggest that in this atherogenic mouse model treated with RSG, lipid metabolism can be compromised during Zn deficiency and that adequate dietary Zn may be considered during therapy with the anti-diabetic medicine RSG [42].

Compared with the above severe Zn deficiency, a few studies also examined whether moderate Zn deficiency is associated with the increase of the risk for cardiovascular diseases. An early study evaluated the association between blood pressure and vascular nitric oxide (NO) pathway using male Wistar rats [43,44]. Weaned male Wistar rats were divided into two groups and fed either moderately Zn-deficient diet (Zn content 8 - 9 mg/kg) or a control diet (Zn content 30 mg/kg) for 60 days. They found that Zn deficiency induced an increase in blood pressure from day 30 of the experimental period, leading to hypertension on day 60. Animals that were fed the Zn-deficient diet had lower urinary excretion levels of nitrates and nitrites and higher intensity of spontaneous luminescence on day 60. They also found that Zn-deficient rats showed a decrease in glomerular filtration rate and no changes in sodium and potassium urinary excretion. Zn deficiency decreased NADPH diaphorase activity in glomeruli and tubular segment of nephrons, and reduced activity of nitric oxide synthase in the renal medulla and cortex. Zn deficiency induced a reduction in nephron number, glomerular capillary area and number of glomerular nuclei in cortical and juxtamedullary areas. Zn deficiency also increased the number of apoptotic cells in distal tubules and cortical collecting ducts neighboring glomeruli and, to a lesser extent, in the glomeruli [44]. Other studies also demonstrated the association between moderate Zn deficiency and the increased risk of cardiovascular diseases [45].

We have recently revealed that Zn deficiency exacerbates diabetes-induced testicular [46,47] and hepatic damage [48]. We have used an animal model in which diabetes was induced by MLD-STZ and some of diabetic mice were treated with TPEN for 4 months to induce systemic Zn deficiency. The diabetic mice with Zn deficiency showed a significant severe damage in the testes, liver, and kidney as compared to the diabetic mice without Zn deficiency. These animal studies supported the finding from a human study with type 2 diabetic patients. In the human study, two groups of diabetic patients, one group of patients with low serum Zn levels and one group of patients with relative high serum Zn, the former exhibited high risk of cardiovascular events as compared to those with relatively higher serum Zn level [49].

Effect of diabetes on Zn homeostasis

Diabetes impairs Zn homeostasis

Terres-Martos et al. examined the status of serum Zn in 18 patients with diabetes and compared it to healthy-age matched controls [50]. Serum Zn concentration was significantly low in diabetic patients as compared to controls. In contrast, copper (Cu) levels were not significantly different compared to controls. This was confirmed by consequent studies [51-53]: the significant Zn deficiency in plasma of diabetic patients. In induced or genetically diabetic animal models, the low level of serum Zn has been often observed relative to controls [54,55]. However, these parallel results could not ensure whether Zn causes diabetes or diabetes affects Zn homeostasis.

Increasing evidence indicates that diabetes can affect Zn homeostasis in many ways, but it is most likely the hyperglycemia, rather than any primary lesion related to diabetes, to cause the increased urinary loss and decrease in total body Zn. Zn excretion through urine is significantly increased in diabetic patients [56-59]. However, whether significantly low level of plasma Zn would be seen in the diabetic patients and animals is still dependent on many factors such as the types of diabetes, duration of diabetes, and the age when diabetes occurs [51-53,56-60].

Quilliot et al. have analyzed the effects of hyperglycemia, malabsorption, and dietary intake on Zn level in 35 men with alcohol-induced chronic pancreatitis complicated by insulin-treated diabetes, 12 men with chronic pancreatitis but no diabetes, 25 men with IDDM, and 20 control subjects [61]. Diabetes due to chronic pancreatitis was associated with decreased plasma Zn concentrations. Of the chronic pancreatitis patients, 17% had low plasma Zn. None of IDDM patients who did not have pancreatitis had low plasma concentrations of Zn. Hyperglycemia, as assessed by fasting plasma glucose and by plasma glycated hemoglobin (HbAlc), was responsible for the increased Zn excretion. The perturbations of Zn metabolism were particularly pronounced in subjects with chronic pancreatitis plus diabetes.

Kechrid et al. performed an experiment on the ⁶⁵Zn turnover under normal and Zn-deficient conditions using genetically diabetic mice (C57BL/KsJdb/db) and non-diabetic heterozygote litter-mates (C57BL/KsJdb/+ ) [40]. They found that pancreatic Zn was lower in the diabetic mice than the non-diabetic mice, although the levels of dietary Zn did not affect the pancreatic levels in both diabetic and non-diabetic mice. Whole-blood glucose concentration was found to be significantly higher in the mice with low-Zn diet. Rate of ⁶⁵Zn loss was similar in both diabetic and non-diabetic mice fed with normal Zn diet, but diabetic mice had a significantly greater whole-body ⁶⁵Zn loss than non-diabetic mice when they both were fed with low-Zn diet. These results suggested that there is almost a dramatic decrease in Zn turnover rate immediately following the introduction of the low Zn diet to animals, but the diabetic mice were less able to reduce Zn loss compared to non-diabetic ones.

In terms of the effects of Zn supplementation on the biokinetics of ⁶⁵Zn in whole body, liver and its biodistribution in diabetic rats, Pathak et al. found that alloxan-induced diabetic rats showed a significant decrease in both the fast and slow components of biological half-life of ⁶⁵Zn which, however, were normalized in whole body following Zn supplementation. In case of liver, slow component was brought back to the normal but the fast component was not increased significantly. The present study indicates that the paucity of Zn in the tissue of the diabetic animals was due to decreased retention of tissue Zn as evidenced by increased serum Zn, hyperzincuria and increased rate of uptake of ⁶⁵Zn by the liver. Zn supplementation caused a significant improvement in the retention of Zn in the tissues and is therefore likely to be of benefit in the treatment of diabetes [62].

Effect of diabetes on Zn exporter and importer

ZnT family regulates Zn fluxes into sub-cellular compartments. It is known that β cells depend on Zn for both insulin crystallization and regulation of cell mass. Therefore, a study has investigated the effect of glucose and Zn chelation on ZnT gene and protein levels and apoptosis in β cells and pancreatic islets, and also the effect of ZnT3 knock-down on insulin secretion in a β cell line and ZnT3 knockout on glucose metabolism in mice during streptozotocin (STZ)-induced β cell damage. They found that in INS-1E cells 2 mmol/L glucose downregulated ZnT3 and upregulated ZnT5 expression, but 16 mmol/L glucose increased ZnT3 and decreased ZnT8 expression, relative to control (5 mmol/L glucose) group. Zn chelation by N,N-diethyldithiocarbamate (DEDTC) lowered INS-1E insulin content and insulin expression. Furthermore, Zn depletion increased ZnT3 and decreased ZnT8 gene expression whereas the amount of ZnT3 protein in the cells was decreased. The most responsive Zn transporter, ZnT3, was investigated further by immunohistochemistry and western blotting: 44% knock-down of ZnT3 by siRNA transfection in INS-1E cells decreased insulin expression and secretion, but STZ-treated mice had higher glucose levels after ZnT3 knockout, particularly in overt diabetic animals. These data suggest that Zn transporting proteins in β cells respond to variations in glucose and Zn levels. ZnT3 gene expression presents in β cells and is upregulated by glucose in a concentration dependent manner or by Zn depletion. However, Zn depletion decreased ZnT3 protein levels [63].

In a recent study, the investigators recruited 75 patients with type 1 or type 2 diabetes and 75 non-diabetic sex-/age-matched control subjects in order to analyze differences concerning human ZnT8 expression, single nucleotide polymorphisms (SNPs) in the genes of ZnT8. Serum Zn was significantly lower in diabetic patients compared to controls, although intracellular Zn showed the same tendency. Interestingly, type 2 diabetes patients treated with insulin displayed lower serum Zn compared to those without insulin treatment. In vitro analyses showed that insulin leads to an increase in intracellular Zn and that insulin signaling was enhanced by elevated intracellular Zn concentrations. They made a conclusion that type 1 and type 2 diabetic patients suffer from Zn deficiency, and Zn supplementation may qualify as a potential treatment adjunct in type 2 diabetes by promoting insulin signaling, especially in Zn-deficient subjects [64].

Since human genetic studies have revealed that common variants of the ZnT8 gene are strongly associated with type 2 diabetes, animal study was done to investigate the expression of ZnT8 in the pancreas and adipose tissue of homozygous db/db mice compared to heterozygous sibling db/+ mice. ZnT8 at both mRNA and protein levels in the pancreas and the epididymal and visceral fat of db/db mice were reduced. These findings suggest that ZnT8 synthesis in the pancreas and adipose tissue is downregulated in db/db mice, and reduced ZnT8 production in the pancreas may advance defects in insulin secretion in diabetes [65].

Factors that affect Zn intake

Phytic acid is a major determinant of Zn bioavailability. A cross-sectional study was done to measure and explore the relationships among phytic acid intake, Zn bioavailability, and molecular markers of Zn homeostasis in 20 type 2 diabetic women compared to 20 healthy women. The phytate/Zn, (calcium+ phytate)/Zn, and (calcium+ magnesium+ phytate)/Zn molar ratios were used to indicate Zn bioavailability. Plasma Zn concentrations and Zn transporter (ZnT1, ZnT8, and Zip1) gene expression in mononuclear cells were measured. Participants with diabetes consumed a similar amount to the intake of healthy women. Bread products and breakfast cereals contributed more than 40% of the phytic acid intake in each group. A positive relationship was observed in all participants between phytic acid and dietary fiber (P<0.001) and between dietary fiber and the (calcium+ phytate)/Zn ratio (P<0.001). Compared to the healthy group, the mRNA ratio of ZnT1 to Zip1 was lower in participants with diabetes, which may indicate perturbed Zn homeostasis in the disorder. These results suggested that the greater amounts of dietary fiber, much of which is associated with phytate, the higher risk of Zn deficiency [66]. However, the increased intake of fiber in the diet often happens in diabetic patients, which needs to be cautious.

Changes of MT with Zn

As mentioned in the section of Introduction, MT plays an important role in Zn and Cu metabolisms. Effects of diabetes on tissue MT expression have been paid attention to by the group of Failla since 1981. First, they examined the MT contents in multiple organs including liver, kidney, intestine, spleen, muscles and plasma of STZ-induced diabetic rats 10 days after STZ treatment [67]. Only hepatic and renal MT contents were significantly increased in diabetic rats as compared to control rats. Furthermore, renal MT mainly bound to Cu, while hepatic MT bound to both Zn and Cu. Later, they extended the study to 21 and 28 days after STZ treatment and found the same trend, i.e., only hepatic and renal MT contents were increased and hepatic MT bound to both Zn and Cu while renal MT mainly bound to Cu. This also implied that renal Zn/Cu ratio increased in diabetic rats as compared to normal rats [68]. They also demonstrated that the increased Zn and Cu levels in both liver and kidney were not associated with the increased absorption of Zn and Cu since no difference for their absorption was found between STZ-diabetic rats and control rats [69]. However, another study seems to find that the Zn and Cu intestinal absorptions were altered [70].

Mechanisms by which diabetes induced MT synthesis vary in different tissue. The hepatic MT synthesis is most likely related to diabetes-related endocrinal imbalance, while renal MT synthesis is more related to increased renal Cu accumulation [71]. Although STZ was found to induce hepatic and renal MT synthesis at a dose-dependent manner [72], the hepatic and renal MT synthesis in STZ-induced diabetic rats was not predominantly secondary to STZ effect. That is because the hepatic and renal MT synthesis along with increases in hepatic and renal Zn and Cu was all preventable by supplementation with insulin to diabetic rats [67]. In addition, Zn and Zn-MT were also significantly increased in the livers of type 2 diabetic animal models, ob/ob diabetic mice [73] and BB rats [74]. Insulin treatment could prevent Zn and Cu from increasing as well as binding to MT [74], suggesting the increased hepatic MT synthesis is predominantly related to chronic endocrine imbalance.

To support above notion, we have demonstrated that hepatic and renal MT proteins were increased in STZ-induced diabetic rats one and six months after diabetes as control rats, along with an increase in both hepatic and renal ET-1 mRNA. The increased hepatic MT protein level was associated with decreases in hepatic Cu and Fe, whereas increased renal MT was associated with increases in renal Cu and Fe accumulation. Zn levels were unaltered in both organs in diabetic rats. Treatment with bosentan, a potent orally active dual ET receptor blocker, partially prevented the increase in MT levels in both liver and kidney. No significant effects of bosentan treatment on non-diabetic rats were observed [75].

Although normally there was no much free Zn, transient increase in intracellular Zn often happens under certain abnormal conditions. Excessive accumulation of intracellular free Zn concentration ([Zn²⁺]i) is cytotoxic. For instance, [Zn²⁺]i can increase rapidly in cardiomyocytes because of mobilization of Zn²⁺ from intracellular stores by reactive oxygen species (ROS), which can directly and/or indirectly damage cardiomyocytes in diabetes. Ayaz and Turan have investigated how elevated [Zn²⁺]i in cardiomyocytes causes diabetes-induced alterations in intracellular free calcium concentration ([Ca²⁺]i) [76]. They used cardiomyocytes from normal rats loaded with fura-2 to fluorometrically measure resting [Zn²⁺]i by fluorescence quenching with the heavy metal chelator TEPN. They showed that diabetic cardiomyocytes exhibited a significant increase of [Zn²⁺]i and [Ca²⁺]i, along with decreases in MT and reduced glutathione, increases in lipid peroxidation and nitric oxide products, and decreased activities of superoxide dismutase, glutathione reductase, and glutathione peroxidase. Treatment of diabetic rats with sodium selenite at 5 µM/kg body weight daily for 4 weeks prevented these defects induced by diabetes [76]. This study suggests that MT that binds Zn in the heart may be quickly oxidized by the overgenerated oxidative stress caused by diabetes, leading to rapid release of Zn from MT, and the suddenly accumulated Zn at high level also cause certain damage to cardiomyocytes.

Zinc supplementation

Zn prevention of insulin resistance, metabolic syndrome and diabetes

The first study on the preventive effect of Zn on alloxan-induced diabetes was performed by Tadros et al., but they used multiple minerals including Zn, Mn, Cr and Co [77]. Yang and Cherian for the first time [78], followed by others, used Zn alone to investigate the prevention of diabetes by Zn supplementation as well as the possible mechanisms. These results are summarized in Table 1. In general, this protection was associated with several fold increases in the concentration of Zn in plasma and pancreas, without any effect on food intake, body weight gain or tissue copper content. The possible mechanisms by which Zn supplementation prevents diabetes include: pancreatic MT induction, Zn anti-inflammation and antioxidant action, and suppression of NF-κB and/or AP1 activation. Supplementation of Zn by subcutaneous or intraperitoneal injection, drinking water and dietary food was all effective in preventing diabetes. Zn supplementation provided the preventive effect on diabetes development in various diabetic models. For instance, a diabetic model induced by multiple low doses of STZ mechanistically was considered to be different from that induced by single high dose of STZ [79-81]. Zn supplementation not only prevented diabetes induced by single dose of STZ or alloxan, but also prevented diabetes induced by multiple low doses of STZ, and even diabetes of genetically pro-diabetic models such as BB Wister rat, NOD and db/db or od/od mouse.

Leptin is thought to be a lipostatic signal that contributes to body weight regulation. Zn might play an important role in appetite regulation and its administration stimulates leptin production. A prospective double-blind, randomized, clinical, placebo-controlled study, with 56 normal glucose-tolerant obese women were randomized for treatment with 30 mg Zn daily for 4 weeks. Baseline values of both groups were similar for age, body mass index, caloric intake, insulin concentration, insulin resistance, and Zn concentration in diet, plasma, urine, and erythrocytes. After 4 weeks, body mass index, fasting glucose, and Zn concentration in plasma and erythrocyte did not change in either group although Zn concentration in the urine increased in the Zn-supplied group (P<0.05). Insulin did not change in the placebo group whereas there was a significant decrease of this hormone in the Zn-supplemented group. The homeostasis model assessment (HOMA) also decreased (P<0.05) in the Zn-supplemented group but did not change in the placebo group [82].

The effect of Zn supplementation on insulin resistance and components of the metabolic syndrome in prepubertal obese children was also investigated recently. There was a report using a triple-masked, randomized, placebo-controlled crossover trial among 60 obese Iranian children. Group 1 participants received 20 mg Zn and group 2 received placebo on a regular daily basis for eight weeks. After a 4-week wash-out period (WO, Fig. 2), the groups were crossed over. After receiving Zn, in either group 1 or group 2, the mean fasting plasma glucose (FPG), insulin and HOMA-estimated insulin resistance (HOMA-IR) decreased significantly, while body mass index, waist circumference, and triglycerides did not significantly change. After receiving placebo, the mean FPG, insulin and HOMA-IR increased significantly, while body mass index, waist circumference, and triglyceride showed a non-significant increase [83], as summarized in Fig. 2.

These results suggest that besides lifestyle modification, Zn supplementation may be considered as a useful and safe additional intervention treatment for improvement of cardiometabolic risk factors related to childhood obesity. However, there was another study that did not support this notion [84]. Forty Korean obese women aged 19 - 28 years were recruited for this study. Twenty women of them took 30 mg/d of supplemental Zn as Zn gluconate for 8 weeks and the rest women took placebo. At the beginning of study, dietary Zn averaged 7.31 mg/d and serum Zn averaged 12.98 µmol/L in the Zn group. Zn supplementation increased serum Zn by 15% and urinary Zn by 56% (P<0.05). HOMA values tended to decrease and insulin sensitivity increased slightly in the Zn group, but not significantly so. No change for other measurements was seen before and after Zn supplementation in either the Zn or control group [84].

Interestingly, type 2 diabetic patients treated with insulin displayed lower serum Zn compared to those without insulin injection. In vitro analyses showed that insulin leads to an increase in intracellular Zn and that insulin signaling was enhanced by elevated intracellular Zn concentrations. In conclusion, type 1 and type 2 diabetic patients suffer from Zn deficiency, and Zn supplementation may qualify as a potential treatment adjunct in type 2 diabetes by promoting insulin signaling, especially in Zn-deficient subjects [64].

The above information is based on specific single study with different case numbers with different conditions. A recent meta-analysis has systematically evaluated the literature on the effect of Zn supplementation on diabetes. The literature search was conducted in PubMed, Web of Science, and SciVerse Scopus. The total number of articles included in that review is 25, which included 3 studies on type 1 diabetes and 22 studies on type 2 diabetes. There were 12 studies comparing the effects of Zn supplementation on fasting blood glucose in patients with type 2 diabetes. The pooled mean difference in fasting blood glucose in Zn supplemented group is 18.13 mg/dl lower than that in placebo groups (P<0.05). 2-h post-prandial blood sugar also shows a significant reduction in 34.87 mg/dl in the Zn treated group as compared to placebo (P<0.05). There were 8 studies comparing the effects of Zn supplementation on lipid parameters in patients with type 2 diabetes. The pooled mean for total cholesterol in Zn supplemented group is lower 32.37 mg/dl than that in placebo groups (P<0.05). Low-density lipoprotein cholesterol in Zn treated group also is significantly lower (11.19 mg/dl, P<0.05) than that in placebo group. Summarized results also showed the beneficial effect of Zn supplementation for these type 2 diabetic patients on systolic and diastolic blood pressures. This first comprehensive systematic review and meta-analysis on the effects of Zn supplementation in patients with diabetes demonstrates that although further studies remain required, Zn supplementation has beneficial effects on glycemic control and promotes healthy lipid parameters [6].

Zn and diabetic complications

Diabetic complications are the consequence of the organ injuries caused by diabetes-related multiple pathogenic factors including hyperglycemia, hyperlipidemia and inflammations, and cellular metabolic abnormalities of each organ under diabetic conditions. Since Zn plays a critical role in many important proteins, enzymes and transcriptional factors, Zn deficiency may lead to these enzymes or proteins dysfunction and, eventually, organ’s dysfunction as complication.

NO is produced by NO synthase (NOS) in many cells and plays important roles in the neuronal, muscular, cardiovascular, and immune systems. Diabetes causes overproduction of peroxynitrite through overproduction of superoxide [85-87]. Peroxynitrite as a strong oxidant is able to release Zn from the Zn-thiolate cluster of endothelial NOS (eNOS) leading to dysfunction of eNOS catalytic activity, i.e., decreasing NO synthesis and increasing superoxide production [88]. This finding may be a very important mechanism by which diabetes causes various cardiovascular diseases. Zn deficiency is accompanied by an increase in oxidative stress, and may accelerate the peroxynitrite-induced eNOS uncoupling process. There was a document indicating that Zn deficiency constitutes a pathogenic risk for cardiovascular system [89]. Zn deficiency as a risk for cardiac injury has been implicated in non-diabetic conditions [90]. More importantly, Zn supplementation prevents heart from isoproterenol- or catecholamine- or adriamycin-induced injury [91-93]. The fact that Zn deficiency exaggerates diabetic osteoporosis [94], diabetic testicular damage [46,47], and hepatic damage (Zhang et al. 2012, in press) supports the importance of Zn in maintaining organ’s normal function. All these data suggest that Zn deficiency in diabetes may be a cause of diabetic complications and Zn supplementation may attenuate and prevent the diabetic complications.

Several studies have explored the possible prevention of Zn supplementation for various diabetic complications (Table 2). The first two clinical studies have observed the systemic Zn and antioxidant status [95,96]. They found that Zn supplementation significantly enhanced diabetes-decreased serum Zn level and antioxidant contents in the blood cells. Gupta et al. have investigated the improvement of the severity of peripheral neuropathy in diabetic patients, in addition to examining the modification of serum Zn status. Diabetic patients with neuropathy who received oral Zn sulfate (660 mg/d for 6 weeks) significantly improved motor nerve conduction velocity, but no improvement was noticed in the patients with autonomic neuropathy with same Zn supplementations [7].

Later a few studies using animal diabetic models also demonstrated the preventive effect of Zn supplementation on diabetes-caused injuries. For instance, Mooradian et al. have investigated the role of Zn status in altered cardiac adenylate cyclase activity in diabetic rats [89]. To determine whether the Zn intake of the animal can account for the altered β-adrenergic receptor activity in the diabetic heart, the β-adrenergic receptor number and isoproterenol-stimulated adenylate cyclase activity were examined in diabetic and control rats maintained on low, normal and high Zn diets for 3 weeks. The isoproterenol-stimulated adenylate cyclase activity was significantly lower in diabetic rats on low Zn diets compared with control diet. The effect of dietary Zn content on isoproterenol-stimulated adenylate cyclase was significant in control rats only. Thus Zn intake appears to be an important determinant of cardiac adenylate cyclase activity level. Additional factors peculiar to the diabetic state are involved in the modulation of β-adrenergic responsiveness of the diabetic heart.

However, contradictory outcomes in human studies regarding Zn supplementation on the complications of diabetes were also reported. For instance, the effect of oral Zn supplementation in patients with type 1 diabetes on metabolic control and Zn blood concentrations was done with 20 patients with type 1 diabetes and 17 controls. All measurements were performed before and after oral Zn supplementation was initiated and continued for 4 months in type 1 diabetic patients. There was no significant difference for HbA1c before and after 4-month supplementation. Zn concentrations in plasma were within the normal range in type 1 diabetic patients before and after supplementation and the control; however, Zn concentrations in erythrocyte presented lower than normal values for all groups. A Zn increase in erythrocyte after supplementation was observed in type 1 diabetic patients. More studies are needed to confirm oral Zn supplementation as nutritional management in diabetes [97]. Therefore, in this pilot study with small cases of patients with a normal rage of serum Zn levels and dietary supplementation of Zn based on food intake there was no much positive outcomes. There was also study with 40 type 2 diabetic patients who were supplemented either with 240 mg/d of Zn as Zn gluconate or with placebo for 3 months. Blood and spot urine samples were taken at baseline, day 3 and 7, month 1, 2 and 3 during supplementation and 1 month after cessation. In these patients, despite significantly higher levels of serum Zn in the treatment group, markers of oxidative damage, levels of hydroxyeicosatetraenoic acid products and vascular indices were unchanged by Zn supplementation during the four-month study period [98]. Again improving the Zn status in patients with type 2 diabetes with normal Zn levels did not have any impact on oxidative damage and vascular function, and such supplementation may not be generally beneficial in these individuals.

From the above individual studies, it is clearly that the outcomes of Zn supplementation on diabetic complications were affected by many factors, including sample size, Zn status before Zn supplementation and the ways of Zn supplementation.

Possible mechanisms of Zn prevention of diabetic complications

Although the exact mechanisms by which Zn supplementation prevents diabetic complications remain unknown, several possibilities have been implicated as discussed in the below sections.

Insulin function

Patients with type 1 diabetes do not produce insulin, and patients with type 2 diabetes has insulin resistance in peripheral tissue; therefore, diabetic complications are attributed to both the toxic effect of hyperglycemia, hyperlipidemia and inflammation on target tissue and the abnormalities of glucose metabolisms in the target tissue due to the insulin defect or insulin resistance. There was a study indicating that Zn deficiency significantly decreased the response of tissue to insulin [55] and the metabolic rate, leading to anorexia [99]. Zn has insulin-like effects on cells, including promotion of both lipogenesis and glucose transport, therefore, Zn supplementation to diabetic patients may stimulate tissue to use glucose and maintain a normal lipid metabolisms and cellular normal function. Simon et al. have demonstrated that Zn supplementation enhanced gastrocnemius insulin receptor concentration and tyrosine kinase activity [100]. Fasting serum glucose concentrations were significantly lower in the Zn-treated diabetic group compared with non-Zn-treated diabetic group. There was a negative correlation between femur Zn and serum glucose concentrations. In addition, Zn supplementation markedly ameliorated the hyperglycemia of diabetic mice along with an increase in the leptin production [101-103]. These studies suggest that Zn is a mediator of leptin production.

Zn acts insulin function through a direct effect on insulin signaling and also through indirect action on insulin-like growth factor (IGF) regulation. Treatment of various cells with Zn significantly increased glucose transport and insulin signaling [104-107]. The effect of Zn on insulin signaling was associated with the stimulating multiple components including phosphoinositide (PI) 3-kinase, tyrosine phosphorylation of the insulin receptor β subunit, tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and serine-473 phosphorylation of Akt. Hence, it appears that Zn can induce an increase in glucose transport into cells and potentiate insulin-induced glucose transport, likely acting through the insulin-signaling pathway.

The insulin-like effects of ionic Zn has been confirmed in isolated rat adipocytes at early stage (1982). Concentrations of Zn²⁺ between 250 and 1000 µmol/L stimulated 3-O-methylglucose transport and glucose metabolism to CO₂, glyceride-fatty acid, and glyceride-glycerol. Selective stimulation of the pentose phosphate cycle was also observed since a Zn²⁺-induced increase in glucose carbon 1 oxidation persisted even when glucose transport was blocked with 50 µmol/L cytochalasin B or when transport was no longer rate-limiting for metabolism at high concentrations of glucose [108].

The stimulation of tissue to use glucose can also be reflected by the fact that Zn supplementation reduced the fasting plasma glucose levels in both obese (ob/ob) and lean (+ /?) mice by 21% and 25%, respectively (P<0.05). Fasting plasma insulin levels were significantly decreased by 42% in obese mice after Zn treatment. In obese mice, Zn supplementation also attenuated the glycemic response by 34% after the glucose load. This suggests that Zn supplementation alleviated the hyperglycemia of ob/ob mice, which may be related to its effect on the enhancement of insulin activity [109].

IGF-1 therapy in diabetes has been proposed, but it has not been used clinically yet due to several unsolved issues [110]. However, Zn can regulate IGF signaling through maintaining IGF-I and IGF-II in an active form by directly regulating IGF-II binding to IGF binding proteins (IGFBPs) and the type 1 IGF receptor (IGF-1R), by preventing secreted IGF-II from binding to IGFBP-3 and IGFBP-5, thus maintaining IGF-II in an “active state,” i.e., readily available for IGF-1R association. In addition, Zn also decreased the affinity of the IGF-2R. In contrast, Zn increased IGF-I, IGF-II and R(3)-IGF-I binding to the IGF-1R by increasing ligand binding affinity. Therefore, a novel mechanism has been proposed that Zn may alter IGF distribution, i.e., Zn acts to increase IGF-1R binding at the expense of IGF binding to soluble IGFBP-5 and the IGF-2R [111,112].

The insulin-like effect of Zn was also reflected by its inactivation of glycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase linked with insulin resistance and type 2 diabetes. Treatment of HEK-293 cells with Zn was reported to enhance glycogen synthase activity and increase the intracellular levels of β-catenin, providing evidence for inhibition of endogenous GSK-3β by Zn. Moreover, Zn ions enhanced glucose uptake 3-fold in isolated mouse adipocytes, an increase similar to activation with saturated concentrations of insulin [113]. Another report indicated that the treatment of cardiac H9c2 cells with ZnCl₂ (10 µmol/L) in the presence of Zn ionophore pyrithione for 20 min significantly enhanced GSK-3β phosphorylation at Ser9, indicating that exogenous Zn can inactivate GSK-3β in H9c2 cells. The effect of Zn on GSK-3β activity was blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 but not by the mammalian target of rapamycin (mTOR) inhibitor rapamycin or the PKC inhibitor chelerythrine, implying that PI3K but not mTOR or PKC accounts for the action of Zn. In support of this interpretation, Zn induced a significant increase in Akt but not mTOR phosphorylation. The PI3K/Akt signaling pathway is thus responsible for the inactivation of GSK-3β by Zn [114,115].

In terms of mechanisms by which Zn ions have an insulin-like effect, the first possible one may be related to the particularly sensitive target of Zn ions, protein tyrosine phosphatase 1B (PTP 1B), a key regulator of the phosphorylation state of the insulin receptor. Tyrosine phosphatases seem to be regulated jointly by insulin-induced redox (hydrogen peroxide) signaling, which results in their oxidative inactivation and by Zn inhibition after oxidative Zn is released from other proteins. In diabetes, the significant oxidative stress and associated changes in Zn metabolism modify the cell’s response and sensitivity to insulin. Zn deficiency activates stress pathways and may result in a loss of tyrosine phosphatase control, thereby causing insulin resistance [116]. The tight inhibition of protein tyrosine phosphatases by Zn is likely responsible for the known insulinomimetic effects of Zn ions, which increase net phosphorylation of the insulin/IGF-1-receptors and activate their signaling cascades. More importantly, not only do extracellular Zn ions affect signal transduction, but growth factors induce cellular Zn fluctuations that are of sufficient magnitude to inhibit protein tyrosine phosphatases [117].

The tumor suppressor PTEN is a putative negative regulator of the phosphatidylinositol 3-kinase/Akt pathway. Treatment with Zn resulted in a significant reduction in levels of PTEN protein in a dose- and time-dependent fashion in a human airway epithelial cell line. This effect of Zn was also observed in normal human airway epithelial cells in primary culture and in rat airway epithelium in vivo. Concomitantly, levels of PTEN mRNA were also significantly reduced by Zn exposure. PTEN phosphatase activity evaluated by measuring Akt phosphorylation decreased after Zn treatment. Pretreatment of the cells with a proteasome inhibitor significantly blocked Zn-induced reduction of PTEN protein as well as the increase in Akt phosphorylation, implicating the involvement of proteasome-mediated PTEN degradation [118].

The important role of Zn in insulin signaling can also be mirrored by a recent study [119]. In this study, the ability of several compounds and related structures to induce insulin-like signal transduction to downstream effectors such as the transcription factor FOXO1a and the key gluconeogenic regulatory enzymes phosphoenolpyruvate carboxykinase and glucose 6-phosphatase (G6Pase) was investigated. Their results indicated that β-thujaplicin, diethyldithiocarbamate (DEDTC) and its clinically-used dimer disulfiram, induce insulin-like dose-dependent effects on signaling to FOXO1a in a manner that is strictly dependent on the presence of Zn ions, as other ions including aluminum, cobalt, copper, lithium and manganese cannot substitute. The most potent compound tested on gluconeogenesis is disulfiram, which in the presence of 10 µmol/L Zn, inhibited both phosphoenolpyruvate carboxykinase and G6Pase with an IC₅₀ of 4 µmol/L. These results demonstrate that metal binding compounds with diverse structures can induce Zn-dependent insulin-like effects on signal transduction and gene expression [119].

Antioxidant

Oxidative stress plays a critical role in the development of diabetic complications; therefore, prevention of diabetes-suppressed systemic antioxidant capacity may be one reason for the prevention of systemic complications of diabetes by Zn supplementation [120]. It has been implicated that TBARS was significantly increased and activities of antioxidant enzymes, SOD, catalase and GSH were significantly decreased in liver of STZ-induced diabetic rats, which suggests that the structural damage to the tissue or complications of diabetes mellitus may be due to oxidative stress [121]. Zn functions as a complex antioxidant through participation in SODs and thioredoxins [122,123], enzymatic and chelator activities, stabilizes cell membranes, and inhibits lipid peroxidation [124]. It protects tissue from ionizing irradiation and ultraviolet light by induction of MT [10].

Anderson et al. documented the potential beneficial antioxidant effects of the individual and combined supplementation of Zn and Cr in people with NIDDM. These results are particularly important in light of the deleterious consequences of oxidative stress in people with diabetes [125]. Furthermore, similar results were found for Zn supplementation only and even improved the antioxidant activity, red blood cell Cu/Zn-SOD and GPx, which was significantly decreased in diabetic subjects [126]. The fact that SOD-overexpressing mice prevent diabetes-associated embryopathy may implicate that Zn supplementation improved the antioxidants including Cu/Zn-SOD may act as the one of mechanisms [123].

Recently we have demonstrated that Zn deficiency exacerbates diabetes-induced testicular and hepatic damage, which was accompanied with a decreased expression and function of Nrf2 [46,48]. Nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or Nrf2, is a transcription factor that in humans is encoded by the NFE2L2 gene [127]. Nrf2 is a master regulator of cellular detoxification response and redox status to protect cells from various oxidative stresses and damages. We have demonstrated its important role in determining the susceptibility of cells or tissue to diabetes-induced oxidative stress and/or damage [128]. Several studies have demonstrated the induction of Nrf2 by Zn [129-131]. In the above section, the important role of Zn in inhibiting Akt negative regulators PTEN and PTP1B have been discussed, therefore, Zn may be required for maintaining Akt function, which in turn inhibits GSK-3β function by phosphorylating it. Phosphorylated GSK-3β will become inactive form that is unable to stimulate Fyn translocation into the nucleus where Fyn causes the exportation of nuclear Nrf2 to cytoplasm. In the cytoplasm Nrf2 will be bound by Keap 1, leading to Nrf2 degradation, as illustrated in Fig. 4.

Improvement of Zn deficiency and increased ratio of Zn/Cu

Zn deficiency does not have to be the status of serum Zn level absolutely lower than normal range since the ratio of Zn to other metals such as Cu or iron, i.e., Zn/Cu or Zn/Fe, also plays an important role in the pathogenesis of insulin resistance, metabolic syndrome, diabetes, and cardiovascular diseases. Using STZ-induced diabetic rats Aguilar et al. [132] determined the influence of the duration of diabetes on the Zn/Cu ratio by three time periods: 7, 21 and 60 days after diabetes onset in the insulin target tissue (liver, adipose tissue, and skeletal muscle). They found that there was a significant decrease in the ratio of Zn/Cu in the liver and adipose tissue, but not in the skeletal muscle. This decrease was diabetes-dependent and also diabetic duration-dependent. Zargar et al. found that although serum Zn was not decreased in NIDDM patients, but Cu levels significantly increased, leading to a decrease in the ratio of Zn/Cu [133]. Ripa et al. and Canatan et al. also found that Zn and the Zn/Cu ratio were lower in the patients with dilated cardiac failure or etiology of essential hypertension compared to normal controls [134,135]. All these studies suggest the potential risk of decreased Zn/Cu ratio for cardiovascular diseases. However, there was also documentation indicating that Zn/Cu ratio was not lower, and even higher in the diabetic patients. Therefore, whether Zn/Cu ratio decreases in diabetic subjects is dependent on multiple factors [132].

Reportedly Zn supplementation significantly corrected the diabetes-induced Zn deficiency [136]. Correct systemic Zn deficiency would help changing diabetes-induced decrease in Zn/Cu ratio. This theory was strongly supported by the fact that diabetes caused Zn deficiency along with Zn-dependent low immune function [137,138] and Zn supplementation can correct plasma Zn levels and Zn/Cu ratio to normal values, and enhance diabetes-decreased CD4 cells [139].

MT induction

MT as potent antioxidant protects cells and tissue from oxidative damage [10,140]. Zn as potent MT inducer prevented cells and tissue from oxidative stress-induced damage [10,141-145]. Using cardiac-specific MT-transgenic (MT-TG) model in which MT mainly binds with Zn, studies from our group [146-148] and other groups [149,150] have shown that MT-TG mice are highly resistant to diabetes-induced cardiac toxicity. These results suggest that MT induction may be one of the reasons responsible for the preventive effect of Zn supplementation on diabetic complications. For the detail regarding the preventive effect of MT on diabetic cardiomyopathy, see the reviews [26,151].

To support above notion, we have demonstrated that supplementation with Zn for 3 months to STZ-induced diabetic mice significantly prevented the development of cardiomyopathy, showed by increases in cardiac morphological and functional abnormalities at 6 months after diabetes onset, as compared to non-Zn-treated diabetic mice. The cardiac MT was found to be significantly increased in Zn-treated mice [152]. By in vitro study, we further confirmed that exposure of cardiac cells to high levels of glucose and lipid significantly cause cytotoxicity; however, the cytotoxicity was significantly prevented by Zn pretreatment in these cells, which was accompanied by a significant MT induction. To define the direct role of MT in this protective action, low-dose cadmium was also used as MT inducer and found to provide similar prevention as did Zn pretreatment [153]. These studies strongly suggest the important role of MT induction in the preventive effect of Zn supplementation to prevent diabetic cardiac toxicity.

In another study, the prevention of diabetes-induced renal pathogenesis was also reported from our group, showing that the renal protection by Zn supplementation against diabetes was accompanied with a significant upregulation of MT in the renal tubules [154]. Our work was supported by a recent study [155], in which 32 Wistar albino male rats were made diabetic model with STZ and treated with and without 30 mg/(kg∙d) Zn as Zn sulfate for 42 days. At the end of the experiment, the authors found that diabetes resulted in degenerative kidney morphological changes. The MT immunoreactivity level was lower and the kidney lipid peroxidation levels were higher in the diabetes group than in the controls. The MT immunoreactivity levels were higher in the renal tubules of the Zn-supplemented diabetic rats as compared to the non-supplemented diabetic group. The Zn and MT concentrations in kidney tissue were higher in the supplemented diabetic group compared to the non-supplemented diabetes group [155]. All these support that Zn has a protective effect against diabetic damage of kidney tissue through stimulation of MT synthesis and regulation of the oxidative stress.

Current issues for Zn supplementation and perspectives

Although it is clear that Zn supplementation will be beneficial for the patients with diabetes to control glucose levels, correct lipid metabolisms and anti-inflammation, and consequently prevent the development of diabetic complications, whether clinics can directly supplement with Zn to diabetic patients remains a question [156]. Several issues are still needed to be investigated since supplementation alone may not reach to the maximal benefit. In addition, in general Zn is less toxic, but excessive Zn intake is also toxic. For instance, excessive Zn intake may cause undesirable elevation of HbA1c [157] and high blood pressure [158]. In addition, we recently have summarized the other toxic effects of chronic supplementation with Zn at large dose levels [159].

Since one of the common toxic effects of chronic supplementation with Zn is Cu deficiency [159], co-application with other compounds such as low levels of Cu may overcome the toxic effects and can avoid resulting in Cu deficiency without effects on other improvements [160-164]. These studies indicated that combination of oral Zn and magnesium supplementation may also provide a better effect than either one only. In regarding the preventive effect of Zn supplementation on pancreatic β cells leading to prevention of diabetes, combination of Zn with other antioxidants was also been suggested [165].

In addition, although Zn insulin-like function has been implicated as early as 1980 [166], its clinical application directly for lowering glucose level has not become true, mainly due to its lower absorption and high-dose and long-term supplementation requirements. Therefore several Zn complexes have been developed to overcome the low absorption, efficiency and long-term retardation in the body. Although many complexes have been documented with or without improvement for these criteria, Table 3 is just briefly to summarize the advances on the development of Zn complexes to be examined. In general, new Zn compounds have certain improvements for the absorption, high efficiency in the hypoglycemic action, and a relatively long duration in the body. Furthermore, different mechanisms by which Zn acts insulin function were also revealed using these compounds. For example, different Zn complex, ZnSO₄, ZP, ZM, and ZT, were found to affect different sites of insulin signaling pathway [1]. ZP complex acts on the insulin receptor and PI3 kinase, which relate to the glucose uptake [2]. ZnSO₄, ZM and ZT complexes affect glucose transporter 4 (GLUT 4), which is involved in the glucose uptake [3]. All four Zn compounds affect the activation of the phosphodiesterase. These results also indicate that the Zn compounds promote the glucose uptake by affecting at least three sites in certain cells, which in turn normalize the blood glucose levels in the experimental diabetic animals [158]. A more recent study using an in vivo model of STZ-induced diabetic rats demonstrates that compound bis(1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate)Zn(II), Zn(dmpp) [2], significantly lowers the blood glucose levels of individuals [167].

Particular attention to Zn supplementation in elder diabetic patients should be given. Aging progression is related to oxidative stress, and the cardiovascular complications of elder diabetic subjects would be more evident. In addition, aged population is also suffering from relative Zn deficiency [168]. Oral supplementation with Zn prevented aging processes [168,169]. This suggests that elder diabetic patients may benefit more from Zn supplementation to prevent cardiovascular complications. To support this hypothesis, Kajanachumpol et al, have already investigated the effects of Zn supplementation on Zn status, Cu status, and lymphocyte subsets in elderly diabetic patients. They demonstrated that Zn supplementation can correct plasma Zn levels to normal values, and enhance the percentage of CD4 cells, indicating that Zn supplementation might be useful to enhance the immune status in these patients [96]. Therefore, Zn supplementation therapy is a promising future since there is not toxic effect in general [51,53].

The urinary excretion of Zn in patients with IDDM is approximately doubled. In the absence of a compensatory mechanism, this hyperzincuria should induce a deficient or marginal Zn status. Therefore, Cunningham et al. have examined parameters of Zn status in plasma and in blood cells with respect to urinary Zn losses and Zn supplementation in 14 IDDM diabetic and 15 non-diabetic patients [170]. Subsequently, 6 IDDM subjects and 7 non-diabetics were supplemented with 50 mg Zn daily for 28 days. Individuals with IDDM displayed the expected hyperzincuria, but had normal blood Zn parameters. Zincuria increased by a similar amount in both groups during supplementation, as did the mononuclear leukocyte Zn content. However, erythrocyte Zn was refractory, so a trend toward lower erythrocyte Zn in IDDM subjects persisted during Zn supplementation. HbA1c increased markedly in the Zn-supplemented IDDM group. Despite their chronic hyperzincuria, individuals with IDDM appear not to be Zn-deficient. Large-dose Zn supplementation increases mononuclear leukocyte Zn and induces an undesirable elevation of HbA1c in all individuals. This is especially disconcerting for those with IDDM, and may reflect an exacerbation of a chronic “Zn diabetes.” These data suggest a potential for toxicity from large-dose Zn supplementation [170].

As mentioned above, Zn has a wide range of dose safely used for humans. Recent two studies reported the safety of Zn in clinical setting again. First patients with spinocerebellar ataxia type 2 (SCA2) have reduced concentrations of Zn in serum and cerebrospinal fluid. To assess the effect and safety of Zn supplementation, a randomized, double-blind, placebo-controlled clinical trial was designed, in which 36 Cuban SCA2 patients randomly received daily either 50 mg ZnSO₄ or placebo, together with neurorehabilitation therapy during 6 months. They found that the Zn-treated group resulted in (1) a significant increase of the Zn levels in the cerebrospinal fluid, (2) a mild decrease in the ataxia scale subscores for gait, posture, stance and dysdiadochocinesia, (3) a reduction of lipid’s oxidative damage, (4)a reduction of saccadic latency when compared with the placebo group. This study demonstrated that the Zn treatment was safe and well tolerated by all subjects [171].

In another double-blind, placebo-controlled, randomized clinical trial, 60 cirrhotic patients were recruited and divided into two groups receiving either long-term Zn supplementation at 50 mg elemental Zn sulfate daily or placebo. Child-Pugh scores and biochemical markers were assessed for both interventional and control groups at the first day and the end (the 90th day) of the interventional period. In the initial evaluation, 53.30% of patients from Zn-treated group had a Child-Pugh score of 5 - 8 and 46.70% of patients had a score of 9 - 12. In the control group 60.00% had a Child-Pugh score of 5 - 8 and 40.00% of patients had scored 9 - 12. After 90 days, the mean Child-Pugh score in the Zn-treated group showed a significant improvement whereas in the control group, the mean Child-Pugh score increased slightly (P = 0.14). Zn supplementation significantly decreased copper levels. They concluded that low dose Zn supplementation could prevent deterioration of clinical status of cirrhosis and prevent excess Cu accumulation in non-alcoholic cirrhotic patients. Zn supplementation produces metabolic effects and trends toward improvements in liver function, hepatic encephalopathy, and nutritional status [172].

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