Journal of Inorganic Biochemistry 120 (2013) 817

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Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Focussed review

Insulino-mimetic and anti-diabetic effects of zinc

George Vardatsikos, Nihar R. Pandey 1, Ashok K. Srivastava
Laboratory of Cellular Signaling, Montreal Diabetes Research Center, Research Center - Centre Hospitalier de l'Universit de Montral (CRCHUM) and Department of Medicine, Universit de Montral, Montral, Qubec, Canada H1W 4A4

a r t i c l e

i n f o

a b s t r a c t
While it has long been known that zinc (Zn) is crucial for the proper growth and maintenance of normal biological functions, Zn has also been shown to exert insulin-mimetic and anti-diabetic effects. These insulin-like properties have been demonstrated in isolated cells, tissues, and different animal models of type 1 and type 2 diabetes. Zn treatment has been found to improve carbohydrate and lipid metabolism in rodent models of diabetes. In isolated cells, it enhances glucose transport, glycogen and lipid synthesis, and inhibits gluconeogenesis and lipolysis. The molecular mechanism responsible for the insulin-like effects of Zn compounds involves the activation of several key components of the insulin signaling pathways, which include the extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3-K)/ protein kinase B/Akt (PKB/Akt) pathways. However, the precise molecular mechanisms by which Zn triggers the activation of these pathways remain to be claried. In this review, we provide a brief history of zinc, and an overview of its insulin-mimetic and anti-diabetic effects, as well as the potential mechanisms by which zinc exerts these effects. 2012 Elsevier Inc. All rights reserved.

Article history: Received 17 August 2012 Received in revised form 26 October 2012 Accepted 26 November 2012 Available online 3 December 2012 Keywords: Zinc Organo-zinc compounds Diabetes Insulin-mimicker PKB IGF-1R

1. Introduction 1.1. Historical aspects of zinc Zinc (Zn) is a group 12 trace element, and is the 23rd most abundant element in the earth's crust [1]. It is the second most common trace metal found in the body, after iron, and is the fourth most used metal in the world, after iron, aluminum and copper [2]. Zn has been used since the time of the ancient Egyptians, in the form of zinc oxide (ZnO), to aide in the healing of wounds and burns [3]. In the western world, the discovery of pure Zn is most often credited to the German chemist Andreas Marggraf, when in 1746, he extracted Zn by heating calamine ores and carbon together [4]. In humans, Zn is found in all tissues and tissue uids. In an average 70 kg male, the total quantity of Zn is estimated to be approximately 2.3 g, making it the most prevalent trace metal found in tissue (only iron is found in higher concentrations in the human body at about 4 g, but is located primarily in blood) [5]. Over 75% of this Zn is located in skeletal muscle and bone tissue [1]. As well, apart from iron, zinc is the only other element for which nutritional requirements have been established [6].
Corresponding author at: CRCHUM Angus Campus, 2901 Rachel Est, suite 304, Montral, Qubec, Canada H1W 4A4. Tel.: + 1 514 890 8000x23604; fax: + 1 514 412 7648. E-mail address: ashok.srivastava@umontreal.ca (A.K. Srivastava). 1 Current address: Centre for Stroke Recovery, Ottawa Hospital Research Institute, Neuroscience, University of Ottawa, Department of Medicine, 451 Smyth Rd., Ottawa, ON, Canada K1H 8M5. 0162-0134/$ see front matter 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2012.11.006

It has been shown that zinc is essential in growth and development [7] and a zinc deciency has been shown to play a role in multiple diseases, such as malabsorption syndrome, sickle cell disease, chronic liver disease and diabetes [8]. Zinc deciency is common in developing nations, yet is also observed in a great number of men and women, from varying economic classes and cultures, suffering from varying clinical conditions, in the United States [9,7]. According to a WHO report as of year 2004, about 30% of the world's population is zinc decient [10], which is most prevalent in children under 5 years of age in developing countries [11]. In North America, overt zinc deciency is uncommon [12]. When zinc deciency does occur, it is usually due to inadequate zinc intake or absorption, increased losses of zinc from the body, or increased requirements for zinc [13,14,7]. The recommended dietary allowance for zinc is 8 mg/day for women and 11 mg/day for men [15]. 2. Chemistry of zinc 2.1. Inorganic zinc compounds In nature, Zn is only present in its divalent state, Zn(II). It is of low to intermediate hardness (malleable at 100150 C), yet is considered a very dense metal, at 7.13 g/cm3, and is therefore called a heavy metal. Being amphoteric, Zn dissolves in both strong alkaline and acidic solutions, and can thus react easily with the latter or other inorganic compounds to form a variety of salts, all of which are non-conducting and non-magnetic and for the most part, white in color, with very few exceptions, such as chromate [16], which changes colors, from yellow to

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bluegreen, depending on its oxidation state. In industry, this metal is mainly used as a protective coating over iron and other metals against corrosion, in a process called galvanizing. This protective effect comes from the element's ease of oxidation, due to the fact that it only has two electrons on its outer most shell. It is also extensively used in the construction industry to form metal alloys, and also as an electrode in dry cell batteries, due to its ability of reducing other metal states [16]. Zinc oxide (ZnO) is one of the oldest known inorganic zinc compounds. It is an odorless, white/grayish colored powder. ZnO is soluble in acids and bases, yet not soluble in water or alcohols. It is widely used as a pigment in paints, as an absorber of ultraviolet light [17], and as a vulcanizing agent in rubber products [18]. Other common Zn compounds, such as zinc chloride (ZnCl2), zinc sulfate (ZnSO4), and zinc nitrate (Zn(NO3)2) are, on the other hand, very soluble in water and alcohols [19]. 2.2. Organic zinc compounds In contrast to inorganic Zn compounds, organo-metallic Zn compounds do not exist naturally in the environment [2]. The latter are synthesized, and are frequently used as topical antibiotics and fungicidal lotions [20]. Organic Zn compounds can be separated into three main groups [21]: Organic Zn halides (R-Zn-X), X being any halogen. Di-organic Zn molecules (R-Zn-R), R being any alkyl or aryl group. Lithium or magnesium Zn compounds (M +R3Zn ), M being either lithium or magnesium. The rst organo-Zn compound ever synthesized was Diethyl zinc (C2H5)2Zn. It was also the rst ever compound to display a sigma bond between a metal and a carbon molecule [22]. It reacts violently with water and catches re with ease when in contact with air, and therefore must be handled under nitrogen. It is for this reason that (C2H5)2Zn is known as a hypergolic fuel, because it ignites on contact with air. These types of compounds are often used to propel rockets. Some common reactions used to obtain organo-Zn compounds are oxidative reactions, like the one used by Frankland and Duppa to produce diethyl zinc [22], halogen zinc exchange reactions, and transmetalation reactions. Many organic zinc compounds are used for their medicinal or biological functions. For example, Zn-etheylene-bis(dithiocarbamate)), also known as Zineb, and Zn-dimethyl-dithiocarbamate, or Ziram, are often used as fungicides to protect crop harvests from disease and deterioration while still in the eld, in storage, and during transport [23,24]. Zinc carbonate, which is a mineral ore of zinc, is often used in to treat skin problems, and zinc caprylate is most often used in ointments as a topical antifungal cream [19,25]. 3. Zinc in biological systems It has been known for more than a century that Zn is crucial to the proper growth and maintenance of biological systems, when Raulin showed for the rst time in 1869 that Zn was indispensable for the growth of the fungus Aspergillus niger [26]. It took almost sixty years after that discovery to prove that Zn was also essential for the growth of higher forms of plants [2729]. In 1934, it was demonstrated for the rst time that Zn was not only essential in plant life, but also in mammalian life as well, when it was shown that Zn deciency caused growth retardation and loss of hair in the rat [30], and in the mouse [31]. Later studies showed that other animal species, including birds, pigs, sheep, cows and dogs, can also suffer from Zn deciency, which causes retardation in growth, loss of hair, deformed nails, testicular atrophy, hyperkeratosis, conjunctivitis, and skin lesions [3236].

It was not until 1961 though, that Zn was assigned a biological role in humans [37]. It was discovered by Prasad et al. that a group of adult males from Iran, who were anemic, yet who also suffered from dwarfism, hepatosplenomegaly and hypogonadism, were not only iron decient, but were also Zn decient. When given Zn containing iron supplements, the anemia was corrected, the hepatosplenomegaly improved, there was growth of pubic hair, and an increase in genitalia size [38]. Sandstead later conrmed that the cause of all the symptoms seen by Prasad in Iran were due to Zn deciency, and not just iron deciency [39]. He showed that Zn supplementation accelerated growth, more than would an iron and protein supplementation alone, in a group of Egyptian patients. As well, Zn supplementation initiated puberty within 7 to 12 weeks of Zn supplementation in these patients, who were already adults, a phenomenon which would come to completion, with full development of secondary sexual characteristics between 12 and 20 weeks [39]. As stated earlier, Zn is found in most tissues of the human body with a predominant distribution in the pancreas, which contains 20 to 30 g/g of tissue, followed by the liver and bone, which contain 40 to 60 g/g of tissue [3]. Zn is found in relatively small quantities in the blood, as compared to intracellular stores. Zn blood plasma concentrations are on average 12.5 M [40], yet can vary from 10.7 M, the lower limit of normal (after a morning fast) [41], to 21.1 M [42] in healthy individuals. At the molecular level, Zn is an important micronutrient, required for over 300 enzymatic reactions [43], and is part of more than 2000 Zn-dependent transcription factors and other proteins [44]. Zinc is present in the cell nucleus, nucleolus, and the chromosomes. It plays a vital role in the synthesis of DNA and RNA, through zinc metalloenzymes such as RNA polymerase [45], reverse transcriptase and transcription factor IIIA [46], and has a stabilizing role in the structure of DNA, RNA and ribosomes [46]. Zn helps to form certain structures in these metalloenzymes, which allow the proper functioning of the latter. The most common structure is the zinc nger domain, in which the zinc ion forms a loop in the polypeptide chain by creating a bridge between cysteine and histidine residues [47]. The zinc nger domain in these enzymes was found to be essential for the binding of eukaryotic regulatory proteins to specic DNA sequences [48]. More recently, Zn nger proteins have also been shown to bind RNA, as well as being involved in proteinprotein interactions, which may help explain why there are Zn ngers that do not bind either DNA or RNA [49,50]. Zn homeostasis is at least, in part, controlled by metallothionein. Metallothioneins (MTs) are small proteins (approx. 6200 Da) rich in cystein, with a very high afnity for divalent heavy metal ions, such as cadmium, mercury, platinum, silver and Zn [51,52]. MTs are primarily found in the liver, pancreas, kidneys and intestinal mucosa. In this sense, they help protect cells and tissues against heavy metal toxicity, facilitate the exchange of metals functioning as antioxidants in tissues, as well as protecting the cell against hydroxyl free radicals, which can cause an oxidative stress induced apoptosis [52,53]. It has been suggested that MTs play an important role in Zn-induced cell proliferation and differentiation [54], since they are over expressed in proliferating tissues, such as regenerating and developing rat livers, and certain types of tumors [5558]. MTs also play a role in metal exchange with Zn-dependant enzymes, such as carbonic anhydrase (CA), a zinc metalloenzyme that catalyzes the reversible conversion between carbon dioxide and the bicarbonate ion, through the hydration of carbon dioxide (CO2) [5961]. CA, and by association, Zn, is therefore involved in vital physiological processes such as respiration, transport of CO2/bicarbonate between lungs and tissues, pH and CO2 equilibrium, ureagenesis, gluconeogenesis, and lipogenesis [6264], the latter two linking Zn to diabetes and its insulino-mimetic effects. In addition to MTs, other zinc transporter proteins are also involved in regulating the cellular zinc levels. These include zinc

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transporters (ZnT, solute carrier, SLC, 30 gene family), and Zrt/Irt-like proteins (Zip, or solute carrier.SLC 39 gene family) [65,66]. Znts transport zinc from the cytoplasm into the lumen of intracellular organelles or to the outside of the cell, whereas Zips transport extracellular and organellar zinc into the cytoplasm [65,66]. It is notable that ZnT8 is highly expressed in insulin containing secretory granules of pancreatic -cells and plays a critical role in transporting zinc from the cytosol into the vesicles of the secretory granules [66,67]. A genome-wide association study demonstrated an association of a series of ZnT8 (specically expressed in the pancreatic -cells) polymorphisms with human type 2 diabetes [68]. There are also reports suggesting that ZnT8 dysregulation may contribute to altered insulin secretion in db/db diabetic mice [69]. In mice, global loss of Znt8 is involved in exacerbating diet-induced obesity and resulting in insulin resistance, and this may be due to the loss of ZnT8 activity in a tissue other than the -cell [70] suggesting that ZnT8, by altering cellular Zn levels, might contribute to developing diabetes. 4. Zinc and diabetes The cellular zinc levels are tightly regulated, and disturbances of zinc homeostasis have been associated with diabetes mellitus, a disease characterized by high blood glucose concentrations as a consequence of decreased secretion or action of insulin [71]. Dysregulation of Zn homeostatic metabolism within the pancreas impairs a multitude of key processes, including glycemic control [72]. Zn dyshomeostasis, both systemically and in the pancreas, plays an intricate role in the pathology of both type 1 and type 2 DM [7375]. Suboptimal Zn status has been suggested to decrease insulin secretion from the pancreas [76]. Severe Zn deciency induces hyperglycemia and hyperinsulinemia [74], directly implicating Zn in systemic glucose regulation. Consistent with a critical role for Zn in this process, individuals with type 1 DM often have low serum Zn concentrations [77]. The fact that Zn is stored and secreted from the pancreas along with insulin [78] and that it is essential for the synthesis and structural stability of insulin [79] prompted investigators to test the insulin-mimetic and anti-diabetic potential of Zn in in vitro systems as well as in animal models of diabetes. The rst report showing that Zn can mimic insulin was presented in 1980, when it was shown that Zn, in the form of zinc chloride (ZnCl2) can mimic insulin in its ability to stimulate lipogenesis in rat adipocytes [80], even though a connection between Zn and diabetes had been made 14 years earlier, when Quarterman et al. demonstrated that Zn decient animals were less sensitive to insulin and concluded that Zn was in some way involved with insulin action [81]. 4.1. Anti-diabetic effects of zinc in animal models of type 1 diabetes In animal studies, subcutaneous Zn injections (10 mg/kg body weight as ZnSO4) were shown to partially prevent hyperglycemia in streptozotocin (STZ)-induced model of diabetes, through the increased activation of MTs acting as an oxygen free radical scavenger, as well as a reductant of lipid peroxidation in the pancreas and liver [82]. Later studies suggested that these effects were not entirely due to the increased activation of MTs, as Zn pre-treatment (110 mg/kg body weight as ZnSO4) of STZ-induced diabetic MT-null mice improved their diabetic state [83] (Table 1). The aforementioned studies were conrmed by Ohly et al., who showed that Zn-enriched drinking water (25 mmol/l for 1 week) upregulated MTs, as well as prevented diabetes in multiple low dose of STZ (MLD-STZ)-induced diabetes [75] (Table 1). The MLD-STZ mice treated with Zn-enriched drinking water had a significant decrease of their blood glucose concentrations, and maintained a blood glucose concentration under the euglycemic threshold of 13.9 mmol/l, while the non-Zn treated MLD-STZ mice had blood glucose concentrations much higher than the euglycemic threshold [75].

In further support of the anti-diabetic effects of Zn, Tobia et al. showed that high Zn (1000 ppm) supplementation dramatically delayed the onset and reduced the severity of diabetes in BioBreed (BB) Wistar rats, an inbred rat strain that spontaneously develops diabetes, as compared to BB rats fed a normal Zn (50 ppm) diet or low Zn (1 ppm) diet [84] (Table 1). They also demonstrated that the normal zinc and low zinc diet groups developed diabetes almost two weeks before the rst high zinc diet rat became diabetic. Rodents from the latter group also displayed improved glucose tolerance, as well as increased insulin release, compared to the animals in the other two groups [84]. 4.2. Anti-diabetic effects of zinc in animal models of type 2 diabetes The anti-diabetic potential of Zn compounds has also been examined in animal models of type 2 diabetes mellitus. As early as 1985, Begin-Heick et al. showed that a high Zn supplementation (1000 mg Zn/Kg diet) for 4 weeks attenuated fasting hyperglycemia and hyperinsulinemia in ob/ob mice [85], a well established model of type 2 diabetes mellitus. In these studies, high Zn supplementation elevated insulin in pancreatic islets, and attenuated the abnormally high insulin secretory response to glucose in isolated pancreatic islets [85] (Table 1). Later studies showed that even lower doses of Zn for 6 weeks were sufcient to decrease fasting hyperinsulinemia and hyperglycemia, as well as in reducing the body weight in db/db mice, another mouse model of type 2 diabetes [86]. More recently, a complex formed between Zn and Cyclo(His-Pro) (CHP), a metabolite of thyrotrophin-releasing hormone or product of peptide or amino acid sources, was studied in the Goto-Kakizaki (GK) rat, a non-obese type 2 diabetes animal model, exhibiting a mild fasting hyperglycemia [87] (Table 1). CHP metabolism is thought to be directly related to glucose metabolism [87], since STZ-diabetic rats exhibited an increase of CHP levels which decreased after insulin treatment [88], suggesting that blood glucose metabolism is related to gut CHP concentrations [89]. The Zn-CHP compound was shown to improve short-term and long-term oral glucose tolerance, as well as decreased insulin resistance, in aged GK rats [87]. This decreased insulin resistance is thought to be due to the supply of new Zn brought into the system through the Zn-CHP complex. There are several lines of evidence that suggests benecial roles of zinc supplementation in humans and animals. However, it is presumed but not clear whether the effect is also directly associated with a change in plasma Zn status. Recently, Wang et al. [90] reported in STZ-induced diabetic Wistar rats that zinc supplementation improves symptoms of diabetes such as polydipsia and increased serum level of high-density lipoprotein cholesterol, indicating that zinc supplementation has a potential benecial effect on diabetic conditions. However they found that zinc levels in liver were increased while there was no change in serum zinc levels. 4.3. Role of zinc in insulin resistance and diabetes in humans Abnormalities in Zn homeostasis have been associated with several diseases, including insulin resistance, metabolic syndrome, type 1 and type 2 diabetes (reviewed in [91]). In fact, several studies have reported relatively lower levels of Zn in obese, insulin resistant and diabetic subjects [92], as well as Zn deciency in type 2 diabetes [9396]. Lower Zn plasma concentrations were also associated with increased risk for coronary artery disease [95] and mortality [97] in diabetics. Lower Zn in drinking water is associated with the risk of developing type 1 diabetes during childhood [98] and has been shown to increase pancreatic damage in type 1 diabetics [79]. Zn supplementation has been shown to ameliorate glycemic control in type 1 and 2 diabetes [91]. In addition Zn supplementation reduced Hb1AC levels, corrected lipid proles and decreased albumin excretion in microalbuminuric type 2 diabetics [99102]. Zn has also been found to lower homocysteine levels in microalbuminuric type 2 diabetics while increasing vitamin B12 and

G. Vardatsikos et al. / Journal of Inorganic Biochemistry 120 (2013) 817 Table 1 Anti-diabetic and insulin-like effects of Zn compounds in in vivo and in vitro systems. Type of Zn compound tested In vivo studies in human subjects Zinc (Zn2+) Oral supplementation 2.710.7 mg/day dietary Zn Obese women Reduction in serum insulin levels and Insulin resistance, as measured by the homeostasis model assessment [103107] Decrease in HbA1C levels, triglycerides, total cholesterol and LDL [100] Decrease in HbA1C levels (al-maroof 20) [101] Decrease in HbA1C levels, total cholesterol and cholesterol/HDL ratio [102] Reduced serum Homocysteine, increased folate and vitamin B12 [103] No benecial effects observed [109] No benecial effects observed [108] Mode of administration Doses Model used in study Major physiological effects of treatment

11

Zinc sulfate (ZnSO4) Zinc sulfate (ZnSO4) Zinc sulfate (ZnSO4)

Oral supplementation Oral supplementation Oral supplementation

660 mg/day for 612 weeks 30 mg/day for 12 weeks 22 mg/day for 16 weeks

Type 1 diabetic patients Type 2 diabetic patients Type 2 diabetic patients

Zinc sulfate (ZnSO4)

Oral supplementation

30 mg/day for 12 weeks

Type 2 diabetic patients

Zinc sulfate (ZnSO4) Zinc gluconate In vivo studies in animal models Zinc-3-hydroxy avone

Oral supplementation Oral supplementation

660 mg/day for 68 weeks 240 mg/day for 12 weeks

Type 2 diabetic patients Type 2 diabetic patients

Oral supplementation

5 mg/kg for 30 days

Zinc (Zn2+)

Drinking water

25 mmol/l for 1 week

Zinc carbonate (ZnCO3)

Incorporated in diet

11000 ppm/2 weeks

Zinc (Zn2+) Zn-Cyclo(His-Pro) (Zn-CHP)

Incorporated in diet Gastric gavage or drinking water Intraperitoneal injection

1000 mg Zn/Kg diet 10 mg zinc/kg body weight or 10 mg zinc/L, for 2 weeks 68.8 M Zn(Mal)2/Kg body weight

Bis-maltolato-Zinc (II) (Zn(Mal)2)

Bis(methylpicolinato)zinc (II) (Zn(mpa)2)

Intraperitoneal injection

Bis (allixinato)Zn(II) (Zn(alx)2)

Intraperitoneal injection

Bis(pyrrolidine-N-dithiocarbamate) Incorporated in diet Zn(II) (Zn(pdc)2)

Zn(mpa)2 (I.P., 25.1 mg/kg weight equivalent to 3.0 mg Zinc/kg body weight); Zn(Mal)2 or Zn(pic)2 (I.P., 4.5 mg Zinc/kg body weight) for 2 weeks Type 2 diabetic KK-Ay mice 4.5 mg (68.8 mol) of the Zn/kg body weight for the rst two days, and then the dose was adjusted to approximately 24.5 mg (30.668.8 mol) of Zn/kg body weight according to the blood glucose level observed for the next 12 consecutive days Type 2 diabetic KK-Ay mice 10 mg Zn/kg body mass for the rst 14 days, and the doses were adjusted to 10 or 15 mg Zn/kg body mass for the following 11 days.

Reduction of blood glucose, Decrease in HbA1C levels, uric acid and creatinine [125] Multiple low-dose STZ Increased levels up metallothionine, (MLD-STZ) injection-induced prevention of MLD-STZ-induced diabetes in c57/BL6 and diabetes, prevention of loss of -cell B6SJL/F1 mice function [75] Congenitally diabetic Lower incidence of diabetes, Higher BioBreed Wistar rats pancreatic and serum insulin levels, decreased serum glucose and triglycerides, Improved glucose tolerance, Decrease in islet inammation [84] ob/ob mice Attenuation of exaggerated insulin secretion [85] Goto-Kakizaki Decreased insulin resistance, decreased serum insulin and glucose, improved oral glucose tolerance [87] Normalization of blood glucose, Type 2 diabetic KK-Ay mice improvement in serum insulin and triglyceride levels, improved oral glucose tolerance [116] Type 2 diabetic KK-Ay mice Normalization of blood glucose, decrease in HbA1C levels, improved glucose tolerance [117]

STZ injection-induced diabetes in rats

Normalization of blood glucose, decrease in HbA1C levels, improved oral glucose tolerance, suppression of obesity in type 2 diabetes, decrease in serum insulin and leptin levels [120]

Di(1-oxy-2-pyridinethiolato)Zn complex (Zn(opt)2)

Incorporated in diet

7.5 mg Zn/kg, for 16 days

Type 2 diabetic KK-Ay mice

Normalization of blood glucose, improved oral glucose tolerance, improvement in renal disturbance related to diabetes, decrease in serum insulin, triglyceride and leptin levels, increase in serum adiponectin levels, decrease in blood pressure [122] Normalization of blood glucose, decrease in HbA1C levels, improved oral glucose tolerance, improvement in renal disturbance related to diabetes, decrease in serum insulin and triglyceride levels, increase in serum adiponectin levels, decrease in blood pressure [124] (continued on next page)

12 Table 1 (continued) Type of Zn compound tested In vitro studies Bis-maltolato-Zinc (II) (Zn(Mal)2) Bis(methylpicolinato)zinc (II) (Zn(mpa)2) Bis (allixinato)Zn(II) (Zn(alx)2) Mode of administration

G. Vardatsikos et al. / Journal of Inorganic Biochemistry 120 (2013) 817

Doses

Model used in study

Major physiological effects of treatment

Incorporation into cells

0.125 mM/3 h

Rat adipocytes

Incorporation into cells 100 M1 mM/30 min. Incorporation into cells 0.0011 mM/30 min Zn(II) complexes

Rat adipocytes Rat adipocytes

Bis(pyrrolidine-N-dithiocarbamate) Incorporation into cells 0.0011 mM/30 min Zn(II) Zn(II) (Zn(pdc)2) complexes Di(1-oxy-2-pyridinethiolato)Zn complex (Zn(opt)2) Bis (allixinato)Zn(II) (Zn(alx)2) Incorporation into cells 0.0011 mM/30 min Zn(opt)2

Rat adipocytes

Rat adipocytes

Incorporation into cells 1050 M Zn for 560 min and 50 M ZnCl2, or 25 M zinc complexes for 5 min

3T3-L1 broblasts

Di(1-oxy-2-pyridinethiolato)Zn complex (Zn(opt)2) Zinc chloride (ZnCl2) Zinc sulfate (ZnSO4)

Incorporation into cells 100 nM25 M Zn(opt)2 and 100 M of Zinc complexes for 560 min. Incorporation into cells Not available Incorporation into cells 10100 M for 560 min

3T3-L1 broblasts

Rat adipocytes CHO cells, A10-VSMC, MCF-7 cells

Inhibition of epinephrine-induced free fatty acid (FFA) release, enhances effects of insulin on FFA [115,116] Inhibition of epinephrine-induced free fatty acid (FFA) release [117] Inhibition of epinephrine-induced free fatty acid (FFA) release, induced glucose-uptake [120] Inhibition of epinephrine-induced free fatty acid (FFA) release, induced glucose-uptake [122] Inhibition of epinephrine-induced free fatty acid (FFA) release, induced glucose-uptake [124] Induction of PKB/Akt phosphorylation, Induction of GLUT4 translocation to membrane, Induction of glucose uptake by cells, Inhibition of epinephrine-induced free fatty acid (FFA) release [182] Induction of PKB/Akt and GSK-3 phosphorylation, Induction of GLUT4 translocation to membrane [133] Stimulation of lipogenesis [80] Induction of PKB/Akt and ERK1/2 phosphorylation [144]

This list does not include all the studies showing anti-diabetic and insulin-like effects of Zinc compounds and only representative references are cited.

folate levels [103]. In obese, non-diabetic subjects, insulin sensitivity was improved without changes in leptin levels [104]; however, no preventive effect against diabetes development could be found in obese women [105], but oxidative stress was reduced by Zn in healthy elderly subjects [106]. In a prospective study performed in 8300 women, Zn at higher doses was able to slightly reduce the risk for type 2 diabetes but the effect was limited to the Zn-decient subgroup [107], and it was concluded that higher Zn may be associated with a slightly lower risk of type 2 diabetes in women [99]. In contrast to these studies showing benecial effects of zinc supplementation on diabetes, recent studies in type 2 diabetic patients showed no difference in insulin, HbA1c levels or glycemic control between placebo and zinc treated groups [108,109]. This study also assessed the effect of zinc supplementation in this diabetic group on oxidative damage and vascular function, since vascular dysfunction is common in advanced stages of diabetes. They found that despite higher serum levels of zinc in the treated group, there was no significant difference in markers of oxidative stress, specically allantoin, an oxidation product of uric acid, and of vascular functions measured [108]. The latest meta-analysis of the effects of zinc supplementation on diabetes, however, indicates that zinc supplementation in diabetic patients improves fasting blood glucose, 2-hour post-prandial blood glucose and HbA1c [110]. Physiological doses of Zinc (20 mg/day) for a period of 2 months have been suggested to be benecial in diabetics. This meta-analysis summarized the data from a total of 1362 patients in 25 studies showing benecial metabolic and clinical effects due to zinc supplementations in patients with diabetes mellitus, improved glycemic control. Jayawardena et al. also show an improvement in lipid prole, decrease in lipid peroxidation, decrease in blood pressure and improved antioxidant prole in diabetics [110]. Contrasting results from different studies suggest that further detailed work is required to establish the potential anti-diabetic effect of Zn in human subjects.

5. Anti-diabetic effects of organic zinc complexes in models of type 2 diabetes In most of the animal studies using Zn as an anti-diabetic molecule, either a high dose of Zn (1000 ppm) [84,111,112] or a long treatment period (up to 8 weeks) [113] was used, both of which may be toxic to the organism being treated. Therefore, prompted by the discovery that Bis-maltolato-oxo-vanadium (BMOV), an organo-vanadium compound, was more potent and was without any signicant toxicity than inorganic vanadium salts in improving hyperglycaemia in STZ-diabetic rats [114], Yoshikawa et al. synthesized Bis-maltolato-Zinc (II) (Zn(Mal)2) [115]. At 500 M, this compound was found to inhibit free fatty acid release from epinephrine-stimulated rat adipocytes to a greater extent than VOSO4 and ZnSO4 [115], suggesting that this compound was not only an insulino-mimetic compound, but also an insulin enhancer [116] (Table 1). Furthermore, animal experiments using insulin-resistant KK-A y type 2 diabetic mice demonstrated that intraperitoneal (i.p.) injection of 68.8 M Zn(Mal)2 /kg body weight for the rst two days decreased blood glucose from above 20 mM to just under 11 mM, which was maintained at that level for two weeks [116] (Table 1). In addition, a two-week treatment resulted in improved serum insulin and triglyceride levels and glucose tolerance test in treated mice, as compared to a non-treated KK-Ay control group, without disturbing renal or hepatic function [116]. Subsequently, several new organic Zn compounds were synthesized and were tested for their anti-diabetic potential. Two such compounds, Bis(picolinato)zinc(II) (Zn(pic)2) and Bis(methylpicolinato) zinc(II) (Zn(mpa)2) were synthesized by Yoshikawa et al. [117] and in a comparative study, Zn(mpa)2 (I.P., 25.1 mg/kg weight equivalent to 3.0 mg Zinc/kg body weight, for 2 weeks) appeared to be more potent than Zn(Mal)2 or Zn(pic)2 (I.P., 4.5 mg Zinc/kg body weight for 2 weeks) in improving hyperglycemia in KK-A y type 2 diabetic mice [116] (Table 1). Zn(mpa)2 also improved cholesterol metabolism and decreased HbA1C, an indicator of glycemic control [118,119] while exhibiting no alteration in hepatic and renal functions [117].

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Since then, many other organic Zn compounds have been synthesized, such as bis (allixinato)Zn(II) (Zn(alx)2) [120], bis(2-aminomethylpyridinato)Zn(II) [121], bis (dimethyl dithiocarbamate)Zn(II) and bis (diethyldithiocarbamate)Zn(II) [122], all of which have exhibited potent insulino-mimetic and insulin-enhancing properties in in vitro as well as in vivo experiments. Not only are these compounds more potent than their predecessors, but also improve complications of diabetes, such as obesity-linked hypertension, hyperleptinemia, as well as adiponectin levels [122], which play a protective role against insulin resistance [123]. More recently, oral administration of the newly synthesized bis(pyrrolidine-N-dithiocarbamate)Zn(II) (Zn(pdc)2) (either IP 1 mg Zinc/day for 4 days or oral administration of 1015 mg Zinc for 25 days), improved glucose tolerance and had a hypoglycemic effect in KK-Ay mice [122] (Table 1). Yoshikawa et al. have also recently synthesized a novel Zn complex, Di(1-oxy-2-pyridinethiolato)Zn complex, (Zn(opt)2) [124], which was shown to stimulate glucose uptake in isolated rat adipocytes. Furthermore, oral administration 7.5 mg Zinc/kg for 16 days of Zn(opt)2 in KK-Ay mice exhibited a more potent glucose lowering response, as well as HbA1C levels, than pioglitazone [124]. Glucose lowering effect of a novel Zn-avanol complex has also been reported in streptozotocin-induced diabetic rat model of type-1 diabetes [125]. In these studies, oral administration of 5 mg/kg/day for 30 days was shown to reduce blood glucose and HbA1c in diabetic animals [125]. This organo-Zn compound also improved hyperinsulinemia in KK-Ay mice [124]. Therefore, organic Zn complexes appeared to be more effective than Zn for the insulino-mimetic and antidiabetic actions. 6. Zinc and insulin signaling cascade The precise mechanism by which Zn and its inorganic/organic complexes improve hyperglycemia and glucose homeostasis in diabetes

remains unclear. However, the ability of Zn compounds to increase glucose transport, glycogen synthesis, and lipogenesis, to inhibit gluconeogenesis and lipolysis, and to modulate key elements of the insulin signaling pathway [126133] have been suggested to contribute to this response. Since activation of insulin-induced signaling cascade is critical to exert an insulin-like effect, it is important to discuss this pathway in detail and to highlight the effect of Zn on these signaling events in in vitro and in vivo systems. The rst event in insulin action involves the binding of insulin to its receptor. The insulin receptor (IR) is composed of two extracellular -subunits and two transmembrane -subunits linked to each other by disulde bonds [134]. The -subunit is located entirely outside of the cell and contains the insulin-binding site, while the intrinsic insulin-regulated tyrosine kinase activity possessing -subunit is the intracellular component of the receptor. Binding of insulin to the -subunit of IR causes a conformational change, leading to an enhanced protein tyrosine kinase (PTK) activity of the -subunit by multi-site tyrosine phosphorylation (reviewed in [135]) (Fig. 1). IR then phosphorylates several scaffolding proteins, including insulin receptor subtrates (IRSs), Src homology collagen (Shc) and adaptor protein with pleckstrin homology (PH) and Src homology 2 (SH2) domains [136]. These phosphorylated scaffolding proteins then bind and activate other signaling proteins to trigger different pathways responsible to mediate insulin action. Zn has been shown to enhance the phosphorylation of IR- subunit in 3T3-L1 broblasts, as well as in rat adipocytes [137], most likely through its inhibitory effect on protein tyrosine phosphatases (PTPases) (Fig. 1 and Section 7.2) and thus mimics insulin signaling pathways. In one insulin-stimulated pathway downstream of IR, the IRS-1/ growth factor receptor binder-2 (Grb-2)/mammalian son of sevenless (mSOS) complex results in the activation of Ras and Raf, serine/threonine kinases. Activated Raf phosphorylates MEK (MAPK kinase),

Fig. 1. The potential mechanism for the insulin-like effects of Zn2+. Zn2+ enhances tyrosine phosphorylation of multiple receptor protein tyrosine kinases (R-PTKs), such as Insulin receptor (IR), Insulin-like growth factor type-1 receptor (IGF-1R), and Epidermal growth factor receptor (EGFR). One of the mechanisms by which this is achieved involve an increased production of reactive oxygen species (ROS), leading to the inhibition of protein tyrosine phosphatase (PTPase) resulting in an increased tyrosine phosphorylation of R-PTK. Activated R-PTKs phosphorylate multiple downstream targets, such as IRSs, leading to the phosphorylation and activation of the MAPK/ERK1/2 and the PI3-K/PKB/AKT signaling pathways. ERK1/2 pathway through the activation of transcription factors contributes to the increased nuclear activity, including gene transcription, cell growth and proliferation. PI3-K/PKB/AKT pathway, through its downstream targets, such as FOXO1, GSK3- and m-TOR signals an increase in glucose uptake, glucose transport, glycogen synthesis, lipogenesis and inhibition of lipolysis and gluconeogenesis.

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which in turn phosphorylates extracellular signal-regulated kinase (ERK1/2) on Thr and Tyr residues located in the activation loop of the kinase (Fig. 1). ERK1/2 then phosphorylates and activates a downstream ribosomal protein kinase, p90 rsk. Both ERK1/2 and p90 rsk can be translocated to the nucleus, where they phosphorylate transcription factors, such as c-Jun, CHOP, CREB and MEF-2 (reviewed in [138]), and contribute to the mitogenic and growth-promoting effects of insulin (reviewed in [139]) (Fig. 1). In the other main pathway, binding of the p85 subunit of Phosphatidylinositol 3-Kinase (PI3-K) to IRS activates the p110 catalytic subunit of PI3-K that catalyses the phosphorylation of phosphatidylinositol (PI) lipids at position 3 of the inositol ring, and generates 3-phosphorylated forms of PI, such as phosphatidylinositol 3, 4, 5 triphosphate (PIP3) and phosphatidylinositol 3, 4 diphosphate (PIP2) [140] (Fig. 1). The formation of PIP2 and PIP3 generates recognition sites for PH domain-containing proteins, principally 3phosphoinositide-dependent kinase 1 (PDK1), which is translocated to the plasma membrane along with Protein Kinase B (PKB), also known as Akt [140,141], and other and related serine/threonine protein kinases, which are responsible for phosphorylating and activating several downstream signaling protein kinases, such as PKB, protein kinase C-zeta (PKC-), and p70 ribosomal S6 kinase (p70 s6k) [142,143] (Fig. 1). Direct stimulation of PI3-K by Zn was shown in HNN8 cells [129] and Swiss 3T3 cells [130]. In experiments using 3T3-L1 broblasts and rat adipocytes, Zn treatment induced the tyrosine phosphorylation of IR but was not able to increase tyrosine phosphorylation of either IRS-1 or IRS-2 [137]. However, in these studies, Zn potentially induced the phosphorylation of PKB in a PI3-K-dependent fashion. These investigations also failed to detect any association between IRS-1 and p85 subunit of PI3-K, suggesting that IRS proteins may not play an important role in Zn-induced PI3K/PKB activation [144] in some cell types. Similarly, Zn-induced activation of PKB was also demonstrated in CHO cells in the absence of any detectable IRS-1 phosphorylation [144]. PKB plays a primordial role in mediating glucose transport, glycogen synthesis, gluconeogenesis, lipogenesis, and protein synthesis, through the following mechanisms: increasing the number of GLUT4 transporters available for glucose uptake [145147], by activation of Glycogen Synthase (GS) via phosphorylation of GSK-3 by PKB on Ser 9 resulting in the inhibition of its catalytic activity, increasing glycogen synthesis [148] (Fig. 1) and by suppressing the transcription of PEPCK and G6Pase, as well as phosphorylation of FOX01, to downregulate gluconeogenesis [149,150] (Fig. 1), and by activation of mTOR (mammalian target of Rapamycin), a 4E-BP1 kinase, which deactivates 4E-BP1, releasing eIF-4E, promoting translation initiation [151] (Fig. 1). Zn has been shown to activate PKB in BEAS-2B human bronchial cells [152], HepG2 cells [132], as well as in 3T3-L1 broblasts and rat adipocytes [137]. In addition, Zn was able to activate PKB/Akt phosphorylation in CHO, A10VSMC and MCF-7 cells [144]. The activated PKB contributes to the regulation of glucose transport, glycogen synthesis, gluconeogenesis, lipogenesis, and protein synthesis, through the activation or inhibition of downstream effectors, as mentioned earlier. Zn-induced GSK-3 and FOX01 phosphorylation has also been shown [132], which may mediate the response of Zn on increased glycogen synthesis by upregulation of GS, and inhibition of gluconeogenesis [153]. In addition to activating the PI3-K/PKB pathway, Zn was also shown to activate ERK1/2 in HT-29 colorectal cancer cells, as well as in human epidermal A431 cells [128,126], which may be responsible for its mitogenic effects in these and other cell types, such as CHO, A10 VSMC, and MCF-7 [144]. Several studies have implicated the role of tyrosine kinases other than IR in mediating some of the effects of Zn on MAPK signaling pathways [127]. These mainly include EGFR and IGF-1R.

7. Receptor tyrosine kinases other than Ir as potential targets of zinc 7.1. Epidermal growth factor receptor (EGFR) The EGFR is a receptor tyrosine kinase that is ubiquitously expressed in a variety of cell types, and is most abundant in epithelial cells and many cancer cells [154156]. It belongs to a family containing three other members (ErbB2, ErbB3, and ErbB4) that undergo homodimerization or heterodimerization to induce autophosphorylation and receptor tyrosine kinase activation in response to ligand binding [155,157]. The EGFR contains an extracellular ligand binding domain, a single transmembrane domain and a cytoplasmic tyrosine kinase autophosphorylation and regulatory domain. Dimerization of the receptor activates the intrinsic tyrosine kinase activity of the intracellular domain at different residues, resulting in the recruitment of the SH-containing domain proteins, which trigger downstream events. The phosphorylation of EGFR on tyrosine 1068 is followed by recruitment of the adaptor protein Grb2, leading to the activation of Ras/ERK1/2 pathway. In aortic A10 vascular smooth muscle cells (A10 VSMC), Zn-induced ERK1/2 and PKB/Akt phosphorylation was blocked by AG1478, a tyrosine kinase inhibitor specic to EGFR, suggesting the implication of an EGFR-dependent pathway in Zn-induced PKB and ERK1/2 phosphorylation in this cell type [144]. Zn has also been shown to induce the tyrosine phosphorylation of EGFR in A431 human epidermal carcinoma cells, B82 mouse lung broblasts, and primary HAE cells [158,159,127]. 7.2. Insulin-like growth factor type 1 Receptor (IGF-1R) IGF-1R is also a receptor tyrosine kinase that is very similar to the IR in structural and functional homology [160]. In fact, in cells that express both receptors, hybrid receptors appear to form readily, yet the biological consequence of these hybrids is not clear [161]. The receptor is a tetramer consisting of 2 extracellular -chains and 2 intracellular -chains. An intracellular tyrosine kinase domain is found in the -chain which is believed to be essential for most of the receptors' biological effects [162]. In response to IGF-1, IGF-1R activation mediates tumor cell proliferation, motility, and protection from apoptosis [160]. Binding of IGF-1 or insulin (at very high, unphysiological concentrations) induces the activation of PTK domain of IGF-1R subunit, activating the autophosphorylation of the receptor (reviewed in [160]). Phosphorylation of adaptor/docking proteins, such as IRS-1 or IRS-2, Shc and Grb2 then takes place [163,164]. A recent study has demonstrated that Zn, in the form of ZnCl2, as able to stimulate the activation of IGF-1R in cardiac cells [165], as measured by phosphorylation of Tyr1135/1136 subunits, tyrosine residues whose phosphorylation is required for IGF-1R activation (reviewed in [160]). This phosphorylation was reversed by pre-treatment of the cells with AG1024, a selective inhibitor of IGF-1R tyrosine kinase [165]. Pre-treatment of cardiac cells by AG1024 also inhibited ZnCl2-induced PKB phosphorylation, demonstrating the requirement of IGF-1R tyrosine kinase in Zn-induced PKB phosphorylation [165]. Zn treatment has been shown to induce IGF-1R- subunit phosphorylation in C6 glioma cells [166]. It was recently shown that either IGF-1R or EGFR may mediate mediate Zn-induced ERK 1/2 and PKB phosphorylation in a cell-specic manner [144]. Pandey et al. demonstrated that pharmacological inhibition of IGF-1R blocked Zn-induced ERK 1/2 and PKB phosphorylation in Chinese hamster ovary (CHO) cells overexpressing a human IR (CHO-IR), yet blockade of EGFR in these cells caused no inhibition of Zn-induced ERK 1/2 and PKB phosphorylation [144]. Moreover, Zn-induced increase in PKB and ERK 1/2 phosphorylation in the CHO-IR cells was not associated with an increased tyrosine phosphorylation of IR- subunit or IRS-1 [144]. Furthermore, Zn was able to induce the phosphorylation of ERK 1/2 and PKB in CHO cells overexpressing a tyrosine kinase decient IR, whereas the effect of insulin was signicantly attenuated

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[144]. In IGF-1R knockout cells, both Zn- and IGF-1-induced ERK 1/2 and PKB phosphorylation was blocked [144]. In addition, the blockade of both IGF-1R and EGFR was able to attenuate Zn-induced ERK 1/2 and PKB phosphorylation in A10 VSMC [144]. Therefore, it is possible that Zn-induced signaling responses may be mediated through the activation of either IR, EGFR and IGF-1R, perhaps in a cell-specic fashion [144]. The precise mechanism by which Zn induces the activation of R-PTK remains obscure; however, Zn has been shown to inhibit the activities of protein tyrosine phosphatases (PTPases) [166168]. As key enzymes in the regulation of tyrosine phosphorylations, proteintyrosine phosphatases are central to the control of cellular signaling and metabolism. Zinc ions are known to inhibit these PTPase enzymes even at picomolar concentrations [169]. PTPases catalyze the dephosphorylation of tyrosyl-phosphorylated proteins and are able to shift the equilibrium of the phosphorylation/dephosphorylation cycle to alter the status of tyrosine phosphorylation of proteins. Thus, by inhibiting the PTPase activity, Zn can potentially increase the tyrosine phosphorylation of IR, IGF-1R, EGFR and enhance their catalytic PTK activity, leading to an upregulation of ERK1/2 and PI3-K/PKB signaling events (Fig. 1). Among these phosphatases, protein tyrosine phosphatase 1B (PTP-1B) negatively regulates insulin sensitivity by dephosphorylating the insulin receptor (IR). PTP-1B is not classied as a zinc enzyme, however, PTP-1B is a particular target for Zinc ions and is strongly inhibited by Zn(II), with log K =7.8 +/ 0.1 [170]. PTP-1B is regulated jointly by insulin-induced redox (hydrogen peroxide) signaling, which results in their oxidative inactivation and by their zinc inhibition after oxidative zinc release from other proteins [171]. Metallothionein (MT) can serve as a source for Zn(II) ions, which makes Zn(II) available for PTP-1B [170]. In diabetes, the signicant oxidative stress and associated changes in zinc metabolism modify the cellular response and sensitivity to insulin. Zinc deciency activates stress pathways and may result in a loss of tyrosine phosphatase control, thereby causing insulin resistance. 8. In vitro effects of zinc on glucose and lipid homeostasis 8.1. Effect on glucose transport The stimulatory effect of Zn on glucose transport has been observed in rat adipocytes [172,173], as well as in 3T3-L1 broblasts and 3T3-L1adipocytes [137]. In most of these studies, high Zn concentrations were required to enhance glucose transport. For example, May and Contoreggi used up to 1000 M Zn (in the form of zinc chloride) to stimulate glucose transport [172]. Ezaki later conrmed these experiments, using 1000 M Zn to stimulate glucose transport in isolated rat adipocytes by a post receptor mechanism, which was 67% the value achieved with 1 nM insulin [173]. Further investigations into the molecular mechanisms of the anti-diabetic effects of Zn showed that Zn activated glucose transport in adipocytes and 3T3-L1 broblasts through activation of PI3K and PKB/Akt [137], and in contrast to earlier studies, it was shown that much lower concentrations of Zn (20 to 50 M) were sufcient to signicantly increase glucose transport in 3T3-L1 adipocytes. Considering that normal Zn serum concentration range at approximately 15 M, concentrations varying from 20 to 50 M may be sufcient to evoke a physiologically-relevant increase in glucose transport [137]. Noteworthy is the fact that in the study, Zn had a greater effect on glucose transport in 3T3-L1 adipocytes than 3T3-L1 broblasts. This is explained by the fact that 3T3-L1 adipocytes predominantly express insulin-sensitive glucose transporter protein type 4 (GLUT4), and 3T3-L1 broblasts express insulin-insensitive GLUT1, showing that Zn acts here as an insulin-mimetic, since insulin-stimulated glucose transport is mediated by GLUT-4 [174,175]. Ilouz et al. also showed that physiological concentrations of Zn (10 M), in the form of ZnCl2, stimulated glucose uptake in GLUT4 expressing isolated mouse adipocytes, to a much greater extent than in C6 rat glioma cells and GP8 rat brain endothelial cells, which both express

GLUT1, and not GLUT4 [176]. They go on to show that physiological concentrations of Zn inhibited glycogen synthase kinase 3 (GSK-3), and suggest that inhibition of GSK-3 by phosphorylation is essential for glucose uptake [176]. 8.2. Effect on glycogen metabolism and gluconeogenesis Another physiological response modulated by Zn is its action on glycogen synthesis. As early as 1985, it was shown that rat livers exposed to Zn salts for 30 days contained twice as much glycogen as those of control rats [177]. Not only was there more glycogen, but the storage of glycogen in the liver of the rats was also accelerated by feeding them Zn, in the form of zinc acetate [177]. More recent studies have shown that Zn, in the form of ZnCl2, phosphorylates GSK-3 at physiological concentrations (15 M) in HEK-293 cells [176]. GSK-3 and Forkhead box protein 01 (FOX01) were also shown to be phosphorylated by ZnSO4 in HepG2 cells. Phosphorylation of GSK-3 inactivates it, causing increased glycogen synthesis by upregulating glycogen synthase [153]. Phosphorylation of FOX01, a transcription factor largely involved in glucose metabolism, renders it inactive and results in its exclusion from the nucleus, causing an inhibition of hepatic glucose production, thereby inhibiting gene expression of gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) [149]. An inhibitory effect of Zn on gluconeogenesis in rat renal cortex slices, as well as rat liver parenchymal cells, has also been demonstrated [178,179]. It may be suggested that Zn-induced activation of PI3K/PKB signaling through GSK-3 and FOX01 contributes to enhanced glycogen synthesis, as well as decreased gluconeogenesis. 8.3. Effect on lipogenesis and lipolysis In addition to their action on glucose metabolism, Zn has been reported to alter lipid metabolism both in vivo and in vitro. As mentioned earlier, Coulston and Dandona showed that Zn, in the form of zinc chloride (ZnCl2) can mimic insulin in its ability to stimulate lipogenesis in rat adipocytes [80]. Later studies by May and Contoreggi showed that Zn was able to inhibit lypolysis in isolated rat adipocytes stimulated with the -adrenergic agent ritodrine [172], albeit at a high concentration (500 M). This effect was thought to be due to Zn-induced H2O2 generation, since exogenous catalase reversed the inhibited lypolysis [172], and since H2O2 had already been shown to mimic a variety of insulins' actions, including lipogenesis and inhibition of lipolysis [180]. It was later demonstrated by Shisheva et al. that ZnCl2 increased the uptake of glucose to lipids in rat adipocytes [181]. They also showed that this event was not due to H2O2 production by ZnCl2 as catalase treatment did not inhibit ZnCl2-induced glucose oxidation and its incorporation into lipids [181]. More recently, insulin mimicking suppression of lipolytic activity was exhibited when both ZnSO4 and Zn(Mal)2 [115], as well as Zn(alx)2 [120] showed a potent inhibition of free fatty acid release from epinephrine-stimulated rat adipocytes. 9. Concluding remarks Zn is an essential trace element required for growth and vital activities from the cellular to whole body levels. Abnormalities in Zn homeostasis are associated with several diseases, including growth retardation and delays in puberty, hyperkeratosis, conjunctivitis, dwarsm and hepatosplenomegaly. Zn deciency has also been linked to insulin resistance, as well as type 1 and type 2 diabetes in humans. Zn has also been shown to exert insulin-mimetic and anti-diabetic effects in various experimental in vitro and in vivo models, and in a limited number of human studies. Zn treatment has been found to improve carbohydrate and lipid metabolism, to enhance glucose transport, glycogen and lipid synthesis, and to inhibit gluconeogenesis and lipolysis in animal models of diabetes and insulin resistance. The molecular mechanisms responsible for the insulin-like

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effects of Zn compounds have been shown to involve internalization and distribution of Zinc to the insulin-sensitive organelles, by its transporters ZnT and ZIP; negative regulation of PTPase activity and in turn the activation of several key components of the insulin signaling pathways including ERK1/2, PI3-K/PKB, activation of receptor and/or non-receptor PTKs, all of which play a critical role in the Zn-induced insulin-like signaling events, in a cell-specic manner. The overall ndings in humans are very limited and inconclusive, suggesting a requirement for indepth focused clinical and experimental studies using some of the new organo-Zn-complexes that have shown benecial effects in animal models of diabetes and possess limited or no toxic effects. Abbreviations CHO Chinese hamster ovary CHO-1018 cells Chinese hamster ovary cells overexpressing a mutant human insulin receptor in which Lys1018 had been replaced by alanine, causing a complete loss of the protein tyrosine kinase activity of the insulin receptor CHO-HIR cells Chinese hamster ovary cells overexpressing a normal human insulin receptor EGF epidermal growth factor EGFR epidermal growth factor receptor ERK1/2 extracellular-signal-regulated kinases 1 and 2 GS glycogen synthase GSK-3 glycogen synthase kinase-3 IGF-1 insulin-like growth factor 1 IGF-1R insulin-like growth factor 1 receptor IR insulin receptor IRS-1 insulin receptor substrate 1 MAPK mitogen-activated protein kinase mTOR mammalian target of Rapamycin PI phosphatidylinositol PI3K phosphatidylinositol 3-kinase PKB protein kinase B PKC- protein kinase C-zeta p70 s6k, p70 ribosomal S6 kinase PTK protein tyrosine kinase R-PTK receptor protein tyrosine kinase VSMC vascular smooth muscle cell Acknowledgments The work in the authors laboratory is supported by the funding from the Canadian Institutes of Health Research (CIHR) operating grant number 67037 to A.K.S. G.V. is the recipient of Ph.D. studentships from the Faculties of Medicine, and Graduate and Post-Doctoral studies, Universit de Montral, the Montreal Diabetes Research Center and the Centre de Recherche du Centre Hospitalier de l'Universit de Montral (CRCHUM). References
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