The antidiabetic agent sodium tungstate activates glycogen synthesis through an insulin receptor-independent pathway.

This work was supported by grants FIS 01/836 (Ministry of Health, Spain) and ERDF 1FD97–0812 (Ministry of Science and Technology, Spain)

Tungstate Reduces the Expression of Gluconeogenic Enzymes in STZ Rats

<div><h3>Aims</h3><p>Oral administration of sodium tungstate has shown hyperglycemia-reducing activity in several animal models of diabetes. We present new insights into the mechanism of action of tungstate.</p> <h3>Methods</h3><p>We studied protein expression and phosphorylation in the liver of STZ rats, a type I diabetes model, treated with sodium tungstate in the drinking water (2 mg/ml) and in primary cultured-hepatocytes, through Western blot and Real Time PCR analysis.</p> <h3>Results</h3><p>Tungstate treatment reduces the expression of gluconeogenic enzymes (PEPCK, G6Pase, and FBPase) and also regulates transcription factors accountable for the control of hepatic metabolism (c-jun, c-fos and PGC1α). Moreover, ERK, p90rsk and GSK3, upstream kinases regulating the expression of c-jun and c-fos, are phosphorylated in response to tungstate. Interestingly, PKB/Akt phosphorylation is not altered by the treatment. Several of these observations were reproduced in isolated rat hepatocytes cultured in the absence of insulin, thereby indicating that those effects of tungstate are insulin-independent.</p> <h3>Conclusions</h3><p>Here we show that treatment with tungstate restores the phosphorylation state of various signaling proteins and changes the expression pattern of metabolic enzymes.</p> </div

Sodium tungstate decreases the phosphorylation of tau through GSK3 inactivation

Tungstate treatment increases the phosphorylation of glycogen synthase kinase-3β (GSK3β) at serine 9, which triggers its inactivation both in cultured neural cells and in vivo. GSK3 phosphorylation is dependent on the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) induced by tungstate. As a consequence of GSK3 inactivation, the phosphorylation of several GSK3-dependent sites of the microtubule-associated protein tau decreases. Tungstate reduces tau phosphorylation only in primed sequences, namely, those prephosphorylated by other kinases before GSK3β modification, which are serines 198, 199, or 202 and threonine 231. The phosphorylation at these sites is involved in reduction of the interaction of tau with microtubules that occurs in Alzheimer's disease.This work was supported by grants from the Spanish CICYT (SAF2003-02697 to J.A., SAF2004-06962 to J.J.G., SAF2003-06018 to R.G.), grants from the Spanish Ministry of Health (C03/08 to J.J.G., G03/212 to R.G.), and an institutional grant to CBMSO from the “Fundación R. Areces.”Peer reviewe

Inhibition of GSK3 dependent tau phosphorylation by metals

One of the main pathological characteristics of Alzheimers disease is the presence in the brain of the patients of an aberrant structure, the paired helical filaments, composed of hyperphosphorylated tau. The level of tau phosphorylation has been correlated with the capacity for tau aggregation. Thus, the mechanism for tau phosphorylation could be important to clarify those pathological features in Alzheimers disease. Tau protein could be modified by different kinases, being GSK3 the one that could modify more sites of that protein. GSK3 activity could be modulate by the presence of metals like magnesium that can be required for the proper function of the kinase, whereas, metals like manganesum or lithium inhibit the activity of the kinase. Many works have been done to study the inhibition of GSK3 by lithium, a specific inhibitor of that kinase. More recently, it has been indicated that sodium tungstate could also inhibit GSK3 through a different mechanism. In this review, we discuss the effect of these two metals, lithium and tungstate, on GSK3 (or tau I kinase) activity.This work was supported by grants from the Spanish and CICYT: SAF2003-02697 to JA; SAF2004-06962 to JJG; SAF2003-06018 to RG; from the Spanish Ministry of Health C03/08 to JJG; G03/212 to RG; FIS 04/0607 to JA; and an institutional grant to CBMSO from the “Fundación R. Areces”.Peer reviewe

Tungstate-induced changes in protein phosphorylation.

<p>Phosphorylation of ERK, p90rsk, PKB and GSK3αβ was analyzed by Western blot using phospho-specific antibodies. Total ERK, total p90rsk, total PKB, total GSK3αβ and GAPDH were used as loading controls. Protein expression and phosphorylation were quantified by densitometry of the corresponding Western blot signal. <b>A</b> The images are representative of 3 controls (Ctrol), 3 controls treated with tungstate (Ctrol+W), 4 diabetic animals (Diab), and 4 diabetic animals treated with tungstate (Diab+W) from two independent experiments. In each experiment n = 6 for Ctrol, n = 7 for Ctrol+W, n = 9 for Diab and n = 14 for Diab+W. 20 µg of protein were loaded per well. Analysis of PEPCK expression in the same experimental groups serves as a control of tungstate action in those samples. <b>B</b> Bar graphs represent relative density (phospholylated versus total; PEPCK versus GAPDH) from Western blot signal (n = 3–8 in each group). Error bars represent S.E.M. *, <i>P<0.05; **, P<0.01;</i> ***, <i>P<0.001</i>.</p

Inhibitory effects of tungstate on gluconeogenesis <i>in vitro</i>.

<p>Total mRNA was extracted from primary cultured rat hepatocytes treated with Dexamethasone 100 nM (Dex), sodium tungstate 1 mM (W) or both (Dex+W) for 18 h as indicated, and Real time-PCR was performed. Tungstate treatment increased the expression of c-jun and c-fos alone and in the presence of dexamethasone, whereas PGC-1α mRNA levels were decreased under these two conditions (<b>A</b>). Conversely, both PEPCK and G6Pase were downregulated after the treatment with tungstate alone (W) or in the presence of dexamethasone (Dex+W) (<b>B</b>). <b>C</b> Glucose output was measured in primary cultured hepatocytes incubated with the gluconeogenic substrates lactate (L) and pyruvate (P), as indicated. Data are representative of three experiments performed in triplicate. Error bars represent S.E.M. *, <i>P<0.05; **, P<0.01;</i> ***, <i>P<0.001</i>.</p

Effects of tungstate on glycemia and insulinemia, and on glycogen content and glycogen synthase activity in liver.

<p>Figures in brackets indicate the number of animals used to calculate the mean for each condition. Values are given as mean ± S.E.M.</p>*<p> <i>, P<0.05;</i></p>**<p> <i>, P<0.01;</i></p>***<p><i>, P<0.001</i> compared with control and</p>††<p> <i>, P<0.01;</i></p>†††<p><i>, P<0.001</i> compared with diabetic animals.</p

Tungstate-induced changes in the expression of transcription factors involved in metabolic control.

<p>Total mRNA was extracted and Real time-PCR analysis was performed in the livers of all the experimental groups. Effects of tungstate on c-jun and c-fos expression (<b>A</b>) and on FoxO-1, HNF-4α, and PGC-1α (<b>B</b>). (n = 6–14 in each group). Error bars represent S.E.M. *, <i>P<0.05; **, P<0.01;</i> ***, <i>P<0.001</i>.</p

Schematic representation of the effects of tungstate.

<p>Treatment of diabetic rats and of cultured hepatocytes with tungstate (W) led to changes in the phosphorylation of signaling proteins and in the expression of transcription factors and gluconeogenic genes. All these changes caused a reduction in hepatic gluconeogenesis, thus contributing to the improvement of the diabetic state.</p