Research article
Compendium of the antidiabetic effects of supranutritional selenate doses. In vivo and in vitro investigations with type II diabetic db/db mice

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Abstract

In recent years, a number of investigations on the antidiabetic effects of supranutritional selenate doses have been carried out. Selenate (selenium oxidation state +VI) was shown to possess regulatory effects on glycolysis, gluconeogenesis and fatty acid metabolism, metabolic pathways which are disturbed in diabetic disorders. An enhanced phosphorylation of single components of the insulin signalling pathway could be shown to be one molecular mechanism responsible for the insulinomimetic properties of selenate. In type II diabetic animals, a reduction of insulin resistance could be shown as an outcome of selenate treatment. The present study with db/db mice was performed to investigate the antidiabetic mechanisms of selenate in type II diabetic animals.

Twenty-one young adult female db/db mice were randomly assigned to three experimental groups (selenium deficient=0Se, selenite-treated group=SeIV and selenate-treated group=SeVI) with seven animals each. Mice of all groups were fed a selenium-deficient diet for 8 weeks. The animals of the groups SeIV and SeVI were supplemented with increasing amounts of sodium selenite or sodium selenate up to 35% of the LD50 in week 8 in addition to the diet by tube feeding.

Selenate treatment reduced insulin resistance significantly and reduced the activity of liver cytosolic protein tyrosine phosphatases (PTPs) as negative regulators of insulin signalling by about 50%. In an in vitro inhibition test selenate (oxidation state +VI) per se did not inhibit PTP activity. In this test, however, selenium compounds of the oxidation state +IV were found to be the actual inhibitors of PTP activity.

Selenate administration in vivo further led to characteristic changes in the selenium-dependent redox system, which could be mimicked in an in vitro assay and provided further evidence for the intermediary formation of SeIV metabolites. The expression of peroxisome proliferator-activated receptor gamma (PPARγ), another important factor in the context of insulin resistance and lipid metabolism, was significantly increased by selenate application. In particular, liver gluconeogenesis and lipid metabolism were influenced strongly by selenate treatment.

In conclusion, our results showed that supranutritional selenate doses influenced two important mechanisms involved in insulin-resistant diabetes, namely, PTPs and PPARγ, which, in turn, can be assumed as being responsible for the changes in intermediary metabolism, e.g., gluconeogenesis and lipid metabolism. The initiation of these mechanisms thereby seems to be coupled to the intermediary formation of the selenium oxidation state +IV (selenite state) from selenate.

Introduction

When taken up at the recommended level (animals: 0.15–0.30 mg Se/kg dietary dry matter; humans: 50–70 μg Se daily), selenium performs its physiological functions in the body of animals and humans as an integral part of the redox-active centre of functional selenoproteins [1], [2], [3], [4], [5]. The detoxification of peroxides, the involvement in the regulation of thyroid hormone metabolism and the participation in the reduction of disulfides and ascorbate are the most important functions fulfilled by the functional selenoproteins, glutathione peroxidase, iodothyronine deiodinase and thioredoxin reductase [6], [7], [8].

In human food, selenium is present in two major forms. Feedstuffs derived from animal sources mainly contain selenium in the form of selenocysteine from functional selenoproteins, whereas selenium from plant-derived foodstuffs is present predominantly as selenomethionine. In trace element supplements, selenium is frequently added in the form of inorganic salt, sodium selenite (selenium oxidation state +IV and sodium selenate +VI). Selenium from various dietary sources is absorbed in the jejunum and in the ileum of mammals. The amino acid derivatives selenomethionine and selenocysteine use the same carriers as their sulphur analogues methionine and cysteine [9]. Selenate uses a sodium-sulphate cotransporter for its absorption, which is driven by the activity of Na+/K+-ATPase at the basolateral enterocyte membrane [10]. In contrast, selenite prior to its absorption partially reacts with glutathione and other thiols in the lumen to form selenotrisulfides, which are presumably taken up into the enterocytes by amino acid transporters. Another part of selenite diffuses through the apical membrane and reacts with thiols in the cytosol of enterocytes [10]. The selenium compounds mentioned above are absorbed, to a high extent (> 85%), from dietary sources, but differences exist in the absorption time. As a result of the upstream selenotrisulfide synthesis, selenite absorption is slower than selenate and selenomethionine absorption [10]. Subsequently, the selenocompounds are released into the blood stream at the basolateral enterocyte membrane and distributed to the various peripheral tissues. The exact transport mechanism for the various selenium compounds is not fully understood yet. Selenomethionine associates with hemoglobin, while selenate and the remaining free selenite were found to be transported with α- and γ-globulins [11], [12]. Thus, orally administered selenite presumably enters the peripheral organs in the form of selenotrisulfides, or it is reduced in the erythrocytes. Selenate is metabolised during and after its unmodified uptake by the peripheral tissues (Fig. 1).

This hypothesis of a distinctly different cellular metabolism for selenite and selenate is supported by an investigation into intermediary selenium metabolites after intravenous injection of rats with both compounds [13], [14]. Selenite was rapidly taken up by red blood cells, reduced in the erythrocytes to the selenide oxidation state −II and delivered to peripheral organs (liver) in an albumin-bound form. In contrast, unmodified selenate could be detected in the bloodstream, and the successive reduction to the oxidation state −II takes place during selenate uptake from plasma to peripheral organs. A fraction of “acid labile selenium” consisting of selenium bound unspecifically to proteins (presumably via the formation of Se-S bonds) could be detected. After intravenous injection with both compounds, the main excretion products detected in urine consisted of the methylated forms of selenium (monomethylselenol and trimethylselenonium ion). Injection of selenite (SeIV) led to a major peak of these methylated metabolites in urine after 0–6 h in comparison to a selenate (SeVI) injection, which showed high metabolite concentrations after 6–12 h. Additionally, unmodified selenate was excreted after selenate injection [14].

Selenomethionine is the only selenium compound that can be incorporated unspecifically into proteins instead of its sulphur analogue methionine. The ongoing cellular metabolism of all selenium compounds requires a step-by-step glutathione-dependent reduction to the selenide oxidation state −II, which is the physiological basis for the incorporation of the trace element into the selenocysteine residue of functional selenoproteins by a cotranslational mechanism [15], [16], [17].

In recent years, a fascinating new physiological aspect has been found for selenate. Selenate administration in supranutritive doses (daily administration of amounts up to the individual LD50 for about 8 weeks) to rats with streptocotozin-induced type I diabetes led to a sustained correction of their diabetic status including the decrease of the elevated blood glucose concentration and considerable changes in the expression of abnormally expressed glycolytic and gluconeogenic marker enzymes [18], [19], [20], [21], [22], [23], [24]. From in vivo experiments and in vitro studies with tissue cultures, it was concluded that enhanced phosphorylation reactions at the β subunit of the insulin receptor and further components of the insulin signalling cascade are responsible for the so-called insulinomimetic properties of selenate [25], [26].

Oral treatment of mice with alloxan-induced type I diabetes with a high dose of selenite (4 mg/kg body weight per day) failed to reduce hyperglycemia in these animals, which seems to be based on differences in the intermediary metabolism of selenite and selenate [27].

Insulinomimetic properties of selenate could also be found in type II diabetic db/db mice. In this animal model featuring severe symptoms of type II diabetes [28], [29], the antidiabetic effect of selenate could be attributed to the reduction of insulin resistance, whereas the in vivo administration of selenite did not result in a significant amelioration of insulin resistance and diabetes [28].

Besides the influence of insulin and therefore of insulin sensitising agents on glucose metabolism, hormones also play a crucial role in fatty acid metabolism.

Peroxisome proliferator-activated receptors (PPARs) are originally transcription factors belonging to the superfamily of nuclear receptors, discussed as acting as master regulators of fatty acid metabolism and displaying an important link between fatty acid metabolism and insulin sensitivity. Three isoforms (α, β and γ) have been described. They act on DNA response elements as heterodimers with the nuclear retinoic acid receptor. Their natural activating ligands are fatty acids and lipid-derived substrates. PPARα is present predominantly in the liver and heart and, to a lesser extent, in skeletal muscle. When activated, it promotes fatty acid oxidation, ketone body synthesis and glucose sparing. Peroxisome proliferator-activated receptor gamma (PPARγ) is considered to be one of the master regulators of adipocyte differentiation. The isoform PPARγ2 is abundantly expressed in mature adipocytes and is elevated in animals with fatty livers.

Thiazolidinediones were developed as antidiabetic drugs acting as synthetic ligands of PPARs. They increase peripheral glucose utilisation and reduce insulin resistance [30], [31]. The whole complex of tissue-specific actions and interactions of PPARs is not yet fully understood. In a study with transgenic mice, animals without liver PPARγ but with adipose tissue developed fat intolerance, increased adiposity, hyperlipidemia and insulin resistance. Thus, it was concluded that liver PPARγ regulates triglyceride homeostasis, contributing to hepatic steatosis, but protecting other tissues from triglyceride accumulation and insulin resistance [32], [33]. Moreover, it was shown that the treatment of db/db mice with thiazolidinediones induced expression in the liver of adipose tissue PPARγ target genes, such as adipocyte FABP [34], which foretells that hepatic lipid accumulation (steatosis) could occur during long-term administration [35], [36].

The present study with young female db/db mice was therefore carried out to investigate the mechanisms by which selenate influences insulin resistance and metabolic pathways in type II diabetic mice.

Section snippets

Animals and diets

Twenty-one young female db/db mice (C57BL/KsOlaHsd-Leprdb) aged 6 weeks with an average body weight of 43.7±2.03 g were obtained from Harlan/Winkelmann (Borchen, Germany). The animals had previously been fed a standard chow for mice containing 0.25 mg selenium as sodium selenite per kilogram diet. The mice were randomly assigned to three groups of seven animals each (selenium deficient=0Se, selenite-treated group=SeIV and selenate-treated group=SeVI) and individually housed in plastic cages

Whole-body insulin sensitivity

Fig. 2 shows the results of the whole-body IST. The blood glucose concentrations after the insulin challenge in the experimental groups are given as a percentage obtained in the initial status before putting the mice on special dietary conditions. Selenate treatment kept insulin sensitivity at a comparable level as in the initial status.

In selenium-deficient mice, initial blood glucose concentration (time: 0 min) before insulin injection was 1.5 to 2 times higher than in the initial status and

Discussion

In the present study, treatment of the db/db mice with supranutritional selenate doses effected an improvement of whole-body insulin sensitivity in comparison to selenium-deficient and selenite-treated mice by maintaining insulin sensitivity on a comparably low level as at the beginning of the trial.

Conclusion

The results of our study with type II diabetic db/db mice give some new insight into the mechanisms by which the administration of supranutritional selenate can influence diabetes and insulin resistance. One mechanism of interest is the inhibition of PTPs by intermediary selenate metabolites. This aspect of an antidiabetic action is closely linked to selenium metabolism, since selenium metabolites in the oxidation state +IV are the actual inhibitors of PTPs and they can be generated only from

Acknowledgment

We thank Dipl. Biol. Sandra Schneider and Prof. Dr. R. Schmidt from the Biotechnical Centre of Giessen University for their advice in the RT-PCR experiments. Further thanks are addressed to our Bachelor and Masteral students Linda Minke and Jenny Schaefer for their help with analyses within the scope of their Bachelor and Masteral theses.

For financial support, we thank the H.W. Schaumann Foundation for Agricultural Sciences (Hamburg, Germany).

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