Vanadium and insulin increase adiponectin production in 3T3-L1 adipocytes.

Both adiponectin, an adipokine secreted by adipocytes, and vanadium compounds, have been extensively shown to enhance insulin sensitivity in vivo and in vitro. In this study we examined whether insulin and vanadyl sulfate (VS) affected adiponectin release and cell content from 3T3-L1 adipocytes, and whether they acted through a similar signaling pathway. Adiponectin cell content, but not release, consistently increased in cells treated with insulin (100 nM) and VS (10 and 50 microM) after 24 h. On the other hand, VS-induced adiponectin release only occurred after 4 days of incubation. The protein kinase B (PKB) inhibitor, NL-71-101, decreased both insulin and VS-induced adiponectin cell content, while neither wortmannin nor LY 294002, inhibitors of phosphatidylinositol 3-kinase (PI3-K), attenuated insulin or VS-induced adiponectin cell content. Furthermore, VS-induced adiponectin accumulation occurred in the presence of AGL2263, an insulin receptor (IR) inhibitor. These studies provide the first evidence that vanadium could exert its insulin sensitizing effects through the stimulation of adiponectin through a PKB-dependent transduction pathway.

Nuclear assembly of UGA decoding complexes on selenoprotein mRNAs: a mechanism for eluding nonsense-mediated decay?

Recoding of UGA from a stop codon to selenocysteine poses a dilemma for the protein translation machinery. In eukaryotes, two factors that are crucial to this recoding process are the mRNA binding protein of the Sec insertion sequence, SBP2, and the specialized elongation factor, EFsec. We sought to determine the subcellular localization of these selenoprotein synthesis factors in mammalian cells and thus gain insight into how selenoprotein mRNAs might circumvent nonsense-mediated decay. Intriguingly, both EFsec and SBP2 localization differed depending on the cell line but significant colocalization of the two proteins was observed in cells where SBP2 levels were detectable. We identify functional nuclear localization and export signals in both proteins, demonstrate that SBP2 undergoes nucleocytoplasmic shuttling, and provide evidence that SBP2 levels and localization may influence EFsec localization. Our results suggest a mechanism for the nuclear assembly of the selenocysteine incorporation machinery that could allow selenoprotein mRNAs to circumvent nonsense-mediated decay, thus providing new insights into the mechanism of selenoprotein translation.

Development of an automated protein-tyrosine phosphatase 1B inhibition assay and the screening of putative insulin-enhancing vanadium(IV) and zinc(II) complexes.

The inhibition of protein-tyrosine phosphatase 1B (PTP1B) is a potential target for treatment of type 2 diabetes. Vanadium and zinc metal coordinated complexes have insulin-enhancing activities, and while vanadium compounds inhibit PTP1B, little is known on the mode of action of zinc compounds. In this study we developed an automated PTP1B inhibition assay that allows for a rapid assessment of the PTP1B inhibition strength of candidate compounds. Synthetic vanadium(IV) and zinc(II) complexes were evaluated: IC50 values for vanadium complexes ranged from 0.06 to 0.8 microM whereas for zinc compounds, values were above 10 microM. Vanadium sulfate, a non-conjugated inorganic salt, had stronger inhibition activity than any of the conjugated metal complexes.

Mice with targeted disruption of the Dio2 gene have cold-induced overexpression of the uncoupling protein 1 gene but fail to increase brown adipose tissue lipogenesis and adaptive thermogenesis.

The Dio2 gene encodes the type 2 deiodinase (D2) that activates thyroxine (T4) to 3,3',5-triiodothyronine (T3), the disruption of which (Dio2(-/-)) results in brown adipose tissue (BAT)-specific hypothyroidism in an otherwise euthyroid animal. In the present studies, cold exposure increased Dio2(-/-) BAT sympathetic stimulation approximately 10-fold (normal approximately 4-fold); as a result, lipolysis, as well as the mRNA levels of uncoupling protein 1, guanosine monophosphate reductase, and peroxisome proliferator-activated receptor gamma coactivator 1, increased well above the levels detected in the cold-exposed wild-type animals. The sustained Dio2(-/-) BAT adrenergic hyperresponse suppressed the three- to fourfold stimulation of BAT lipogenesis normally seen after 24-48 h in the cold. Pharmacological suppression of lipogenesis with betabeta'-methyl-substituted alpha-omega-dicarboxylic acids of C14-C18 in wild-type animals also impaired adaptive thermogenesis in the BAT. These data constitute the first evidence that reduced adrenergic responsiveness does not limit cold-induced adaptive thermogenesis. Instead, the resulting compensatory hyperadrenergic stimulation prevents the otherwise normal stimulation in BAT lipogenesis during cold exposure, rapidly exhausting the availability of fatty acids. The latter is the preponderant determinant of the impaired adaptive thermogenesis and hypothermia in cold-exposed Dio2(-/-) mice.

Ubc6p and ubc7p are required for normal and substrate-induced endoplasmic reticulum-associated degradation of the human selenoprotein type 2 iodothyronine monodeiodinase.

The type 2 monodeiodinase (D2) is an endoplasmic reticulum-resident membrane selenoprotein responsible for catalyzing the first step in thyroid hormone action, T(4) deiodination to T(3). Its short half-life is due to ubiquitination and proteolysis by proteasomes, a mechanism that is accelerated by D2 interaction with T(4). To identify proteins involved in D2 ubiquitination, a FLAG-tagged selenocystine133-to-Cys mutation of the human D2 (CysD2) was created and expressed in Saccharomyces cerevisiae using the GAL1 gene promoter. CysD2 activity was detected in the microsomes, indistinguishable from transiently expressed CysD2 in vertebrate cells. Treatment with 100 mg/ml cycloheximide or 30 micro M T(4) caused rapid loss of CysD2 (t(1/2) = approximately 30 min). Clasto-lactacystin beta-lactone not only increased galactose-inducible CysD2 but also stabilized CysD2 in the presence of cycloheximide or T(4). Immunoprecipitation with anti-FLAG antibody combined with Western analysis with antiubiquitin revealed that CysD2 is heavily ubiquitinated. Expression of CysD2 in yeast strains that lack the ubiquitin conjugases Ubc6p or Ubc7p stabilized CysD2 half-life by markedly reducing CysD2 ubiquitination, whereas no difference was detected in Ubc1p-deficient mutants. Similarly, expression of CysD2 in UBC6 and UBC7 mutants also impaired the substrate-induced loss of CysD2 activity and protein. In conclusion, Ubc6p and Ubc7p are required for normal and substrate-induced ubiquitination and proteolysis of D2.