Diabetic mellitus, a chronic metabolic disorder, is one of the most important health problems in the world, especially in developing countries. It is characterized by imperfection in insulin secretion and insulin receptor or post receptor events with derangement in carbohydrate, protein and lipid metabolism and results in chronic hyperglycemia.⁽¹⁾ Chronic hyperglycemia, promotes glucotoxicity, and has negative impacts on various organs and tissues, such as pancreas, liver, kidneys, muscles, etc. Glucotoxicity eventually leads to progressive β-cell dysfunction, impaired insulin gene transcription and permanent β-cell loss due to apoptosis, ensuing in a vicious cycle with exasperation of the hyperglycemia.⁽²⁾ Indeed, hyperglycemia is contemplated to generate reactive oxygen species through diverse pathways, such as mutilation of the redox equilibrium, augmentation of advanced glycation products, activation of protein kinase C or overproduction of mitochondrial superoxides that eventually leading to oxidative stress in a variety of tissues.⁽³⁾ The vulnerability of each tissue to oxidative stress can vary depending upon their expressed antioxidant enzymes. In addition to the pancreatic β-cells, supraphysiological glucose is notorious to provoke oxidative stress in hepatocytes which can cause hepatic tissue damages.^(4,5)

Dietary components can modify the redox status of an organism and appear as an important factor in controlling diabetes and its complications. Nutrients such as iron have a dual role, with the ability to increase or decrease the organism’s redox capacity. Iron, acting as a cofactor for many proteins, including antioxidant enzymes, is essential for cellular metabolism and aerobic respiration. However, in high concentrations it can lead to cellular toxicity and oxidative damage of cellular components due to its role in the formation of free radicals.^(6,7)

Association between excessive systemic iron and diabetes was suggested by the observation that the incidence of diabetes is increased in classical hereditary hemochromatosis,⁽⁸⁾ in which there is a large increase in iron pools. Epidemiological data show a positive correlation between body iron pools and development of glucose intolerance seen in both diabetes type 2^(9–11) and gestational diabetes.⁽¹²⁾ Recently, have been demonstrated, in a mouse model of hereditary hemochromatosis, that glucose uptake is increase in skeletal muscle but glucose oxidation is decreased and the ratio of fatty acid to glucose oxidation is increased, that may to contribute to the risk of diabetes with excessive tissue iron.⁽¹³⁾

Together, these observations suggest that there is a close relationship between iron and diabetes pathophysiology. However, not much data exist regarding this relationship in experimental models. Such models would allow better understanding of the mechanisms involved in this interaction and could contribute to a better control of diabetes and to a reduction of its complications when associated to iron supplementation.

To expand knowledge in between diabetes and iron, we sought to describe the effect of iron and diabetes on oxidative stress, liver histology, and lipid homeostase by studying the effects on peroxisome proliferator-activated receptor-α (PPAR-α) mRNA in liver of hamsters with a diet supplemented with carbonyl iron, an iron for oral use. A growing body of evidence supports the role of PPAR-α in the development of liver disease and as a putative target for the treatment of steatohepatitis. Disabling the PPAR-α gene is known to increase hepatic triglyceride accumulation, especially under conditions of fasting.^(14–16) Pharmacological PPAR-α activation has been shown to lower hepatic triglyceride levels and effectively attenuate steatohepatitis.^(17–19) Furthermore, diabetes can modulate PPARs through increased inflammatory cytokines and oxidative stress in the heart.⁽²⁰⁾ Thus it is possible to suppose that the interaction between iron and diabetes might modulate the PPAR-α expression in the liver and, as a consequence, the lipid homeostasis.

We also studied histologic caracteristics of pancreas sections. This study allowed us to analyze possible iron effects without excessive iron overload by using the hamster as a model, because this animal has a lipoprotein profile similar to that of humans. This allowed us to establish parallels between the alterations seen here and those known to occur in humans, making this a useful model to unravel the mechanisms involved in this relationship. This relationship is especially relevant in developing countries, which have an increasing incidence of diabetes as well as iron deficiency anemia⁽²¹⁾ and, therefore, where an indiscriminate iron supplementation occurs.

Iron treatment increased serum and tissue iron levels. Surprisingly, although both control and diabetics groups have received the same treatment with carbonyl iron, apparently, animals that received both treatments presented an alteration in iron homeostasis, leading to a lower concentration of iron in serum and liver. It is known that the amount of plasma iron is determined by a regulated liberation of iron from most cells of the body. Macrophages, intestinal enterocytes, and hepatocytes have a particularly important role in this process, and this cellular efflux is modulated by liver hepcidin.⁽³⁰⁾ Fernandez Real et al.⁽³¹⁾ verified that hepcidin levels increased significantly in patients with type 2 diabetes. Our data suggest that the interaction between iron supplementation and diabetes triggers changes in iron homeostasis, contributing to a lower absorption, resulting in lower iron levels in serum and liver. This effect can also be attributed to insulin since in vitro data suggest that it is capable of redistributing the cellular pool of transferring receptors, increasing the proportion at the cell surface, leading to increased cellular iron uptake in adipose tissue and the liver.⁽³²⁾ In our experimental model STZ destroys the beta cells, promoting insulin deficiency, which may have caused a smaller deposit of iron in the DI group as compared to CI. Our data on glycemic and lipids profile, besides the histological aspect of the pancreas showed a detrimental interaction between diabetes and iron. These data support those from epidemiological studies which show a correlation between increased iron pools and diabetes. On the other hand, the histological aspect of the liver in group DI followed the same profile of D group and the liver antioxidant levels were increased as compared to group D. Our results show the different tissue vulnerability to ferro-diabetes interaction. Damage through reactive oxygen species is determined not only by the generation of free oxygen radicals but also by the antioxidant defense status of the cell. This is why different organs can exhibit considerable differences in their susceptibility towards cytotoxic damage. Lenzen et al.⁽³³⁾ showed that in pancreatic islets very low levels of gene expression of all antioxidant enzymes are observe compared to liver. Further, seem that the higher expression of PPAR-α and lower oxidative stress played an important role in hepatic integrity. The histological data obtained here indicate that our model of iron supplementation caused less damage to the liver, as we did not see any fibrosis, thereby suggesting preservation of the metabolic functions, what was further supported by total protein and albumin concentrations. When we consider the activity of ALT, a marker of liver function, the DI group showed intermediate levels between C and D. This corroborates with data of oxidative stress and PPAR-α expression in the liver, indicating that the interaction of iron with diabetes reversed damage caused by the disease. As for AST activity, it was shown to be increased in the animals that received iron supplementation, whether they were diabetic or not. An increase in the concentration of AST may reflect damage to other organs such as muscle, since it is not distributed in various tissues. The data of activity of these enzymes also allow us to suppose that the interaction between iron and diabetes promoted different effects on the different tissues, possibly because the susceptibility of these tissues to oxidative stress is different.

In the present study we have found differential expression of liver PPAR-α among diabetic and iron-induced diabetic hamsters. PPAR-α was expressed at relatively lower levels in the diabetic animals. Previous studies have shown that the expression of PPAR-α can be regulated by diabetes. Marcill et al.⁽³⁴⁾ showed that oxidative stress decreases PPAR-α expression in macrophages of diabetics animals. Furthermore, Wang et al.⁽³⁵⁾ investigated the expression of PPAR-α in aorta, renal cortex and retina of diabetic rats and found it to be decreased in all studied tissues, besides Li et al.⁽³⁶⁾ having shown that type 2 diabetic hamsters also had decreased expression of this mRNA in the liver. It has been previously indicated that oxidative stress increases during diabetes^(37,38) and contributes to down-regulation of PPAR-α in atrial myocytes both in vitro⁽³⁹⁾ and in vivo.⁽²⁰⁾ In the present study, diabetes significantly increased oxidative stress as evaluated by increasing carbonyl protein and decreasing glutathione levels and catalase activity, but was attenuated in iron-treated diabetic hamsters. The effectiveness of iron-diabetes to attenuate oxidative stress might be due to the greater induction of NF-E2-related factor 2 (Nrf2) target genes. This activation induces the production of antioxidant compounds and enzymes.^(40,41) Tanaka et al.⁽⁴²⁾ have demonstrated that coordinated induction of Nrf2 target genes protects against iron nitrilotriacetate (FeNTA)-induced nephrotoxicity. Our results concerning antioxidants suggest that the interaction between diabetes and iron alters the redox balance and accounts for increased antioxidant levels, thus contributing to a higher expression of PPAR-α mRNA.

Reduced expressions of PPAR-α have been associated with lipid accumulation.⁽⁴³⁾ Increased concentration of triglycerides and reduced HDL levels are key characteristics of dyslipidemia in diabetes.⁽⁴⁴⁾ Previous studies have shown that excess iron can increase the concentration of triglycerides in the circulation.⁽⁴⁵⁾ Although increases in LDL-cholesterol and triglycerides are common in diabetic dyslipidemia, we did not observe changes in the levels of these metabolites in our diabetes model, probably due to the fact that in STZ-induced diabetes there is a reduction in insulin secretion.⁽⁴⁶⁾ When iron was associated with diabetes, lower cholesterol and higher triglyceride levels were observed. Our data suggest that the alterations in serum lipid homeostasis in group DI are independent of PPAR-α expression because the levels of expression of this mRNA were comparable to the group C and higher than the D groups; this was possibly due to the strong influence of insulin deficiency. HMG-CoA reductase, a key enzyme in the synthesis of cholesterol, has its activity stimulated by this hormone, thus lower insulin levels would lead to less cholesterol synthesis, justifying our data for cholesterol. Lower activity of lipoprotein lipase can contribute to higher levels of triglycerides because this enzyme is essential to the removal of triglycerides from the blood stream and further degradation and it is attenuated in insulin deprivation. Furthermore, although there is no significant difference between body weight of animals in groups D and DI, there is a tendency of group DI to have lower body weight than group D. When we analyzed glucose levels of these two groups it was observed that they were higher in the DI group, suggesting a reduced production of insulin, which can lead to increased lipolysis and proteolysis in this group, leading to greater weight loss in the DI animals as compared to the DI group.

Our data support those from epidemiological studies which show a correlation between increased iron pools and diabetes. It has been reported in clinical studies that increased iron levels in serum and specific tissues may increase the incidence of type 2 diabetes^(32,47) and gestational diabetes.⁽⁴⁸⁾ Iron depletion has been demonstrated to be beneficial in coronary artery responses, endothelial dysfunction, insulin secretion, insulin action, and metabolic control in type 2 diabetes.⁽⁴⁹⁾ Our results show that iron supplemented in diabetes type I model increased serum concentrations of glucose. The interaction between iron and diabetes promoted different effects on the liver and pancreas, possibly because the susceptibility of these tissues to oxidative stress is different. Besides, for the first time, we have shown that PPAR-α expression is differentially expressed in diabetic and iron supplemented-diabetic hamsters livers.

This study was supported by the Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG, Minas Gerais, Brazil n CDS-APQ 01126-08) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil; no. 475823/2007-9).

We thank Dr. Maria Terezinha Bahia (Chagas’ Disease Laboratory, Federal University of Ouro Preto) for the use of the real time PCR ABI 7300 equipment (Applied Biosystems). We are grateful to Rinaldo Cardoso dos Santos for suggestions and careful review of the manuscript.