Rottlerin inhibits multiple steps involved in insulin-induced glucose uptake in 3T3-L1 adipocytes.

Recently, it was shown that rottlerin inhibits insulin-stimulated glucose uptake and reduces intracellular adenosine triphosphate (ATP) levels in 3T3-L1 adipocytes, suggesting that these two events are causally linked. However, several other reports show that ATP-depletion induces glucose uptake in both muscle cells and adipocytes. In the present study, the mechanism of inhibition by rottlerin was studied in detail, in order to resolve this apparent discrepancy. It was found that rottlerin strongly reduces insulin-stimulated 2-deoxyglucose (2-DOG) uptake in 3T3-L1 adipocytes by a partial inhibition of the translocation of the insulin-responsive GLUT4 glucose transporter towards the plasma membrane (PM). Whereas the insulin-induced phosphatidyl-inositol-3' (PI-3') kinase signaling pathway is unaffected by rottlerin, Cbl tyrosine phosphorylation, which provides an essential, PI-3' kinase-independent signal towards GLUT4 translocation, is markedly attenuated. Furthermore, we also observed a direct inhibitory effect of rottlerin on insulin-induced glucose uptake in 3T3-L1 adipocytes. The direct inhibition of insulin-stimulated 2-DOG uptake by rottlerin displayed characteristics of uncompetitive inhibition: with the K(m(app)) of glucose uptake reduced from 1.6 to 0.9 mM and the V(max(app)) reduced from 5.2 to 1.0 nmol/minmg in the presence of rottlerin. In conclusion, rottlerin inhibits multiple steps involved in insulin-stimulated 2-DOG uptake in 3T3-L1 adipocytes. The observed reduction in GLUT4 translocation towards the PM and the uncompetitive inhibition of the glucose transport process provide alternative explanations for the inhibitory effects of rottlerin aside from the effects of rottlerin on intracellular levels of ATP.

Biomechanical behavior of a biomimetic artificial intervertebral disc.

STUDY DESIGN: The biomechanical behavior of a biomimetic artificial intervertebral disc (AID) was characterized in vitro in axial compression and compared with natural disc behavior. OBJECTIVE: To evaluate the strength and durability of a novel biomimetic AID and to demonstrate whether its axial deformation behavior is similar to that of a natural disc. SUMMARY OF BACKGROUND DATA: Current clinically used AIDs have reasonable success rates. However, because of their nonphysiological design, spinal mechanics are altered. To avoid long-term complications, a novel biomimetic AID, with a nucleus-annulus structure and osmotic swelling properties has been developed. METHODS: Eighteen AIDs in total were tested in axial compression. Six were loaded monotonically to determine strength. Six were tested in fatigue (600-6000 N). Another 6 were used to characterize the axial creep and dynamic behavior (0.01-10 Hz). Creep and dynamic response were also determined for 4 AIDs after fatigue loading. RESULTS: The AIDs remained intact up to 15 kN and 10 million cycles. The creep and dynamic behavior were similar to the natural disc behavior, except for hysteresis, which was 20% to 30% higher. After fatigue, creep decreased from 4% to 1%, stiffness increased 2-fold, and hysteresis was reduced to that for a normal disc. CONCLUSION: A strong and durable AID design was introduced. Compared with current clinical articulating AIDs, this biomimetic AID introduces the natural disc annulus-nucleus structure, resulting in axial behavior closer to that of the natural disc.

Design of next generation total disk replacements.

To improve the treatments for low back pain, new designs of total disk replacement have been proposed. The question is how well these designs can act as a functional replacement of the intervertebral disk. Four finite element models were made, for four different design concepts, to determine how well they can mimic the physiological intervertebral disk mechanical function. The four designs were a homogenous elastomer, a multi-stiffness elastomer, an elastomer with fiber jacket, and a hydrogel with fiber jacket. The best material properties of the four models were determined by optimizing the model behavior to match the behavior of the intervertebral disk in flexion-extension, axial rotation, and lateral bending. It was shown that neither a homogeneous elastomer nor a multi-stiffness elastomer could mimic the non-linear behavior within the physiological range of motion. Including a fiber jacket around an elastomer allowed for physiological motion in all degrees of freedom. Replacing the elastomer by a hydrogel yielded similar good behavior. Mimicking the non-linear behavior of the intervertebral disk, in the physiological range of motion is essential in maintaining and restoring spinal motion and in protecting surrounding tissues like the facet joints or adjacent segments. This was accomplished with designs mimicking the function of the annulus fibrosus.

Fourth human parechovirus serotype.

We identified a novel human parechovirus (HPeV) type (K251176-02) from a neonate with fever. Analysis of the complete genome showed K251176-02 to be a new HPeV genotype. Since K251176-02 could not be neutralized with antibodies against known HPeV serotypes 1-3, it should be classified as a fourth HPeV serotype.

Genistein directly inhibits GLUT4-mediated glucose uptake in 3T3-L1 adipocytes.

The isoflavone-derivative genistein is commonly applied as an inhibitor of tyrosine kinases. In this report we analyze the effect of genistein on insulin-stimulated glucose uptake in 3T3-L1 adipocytes. In these cells insulin-induced glucose uptake is primarily mediated by the GLUT4 glucose transporter. We observed that pre-treatment with genistein did not affect insulin-induced tyrosine kinase activity of the insulin receptor or activation of protein kinase B. On the other hand, genistein acted as a direct inhibitor of insulin-induced glucose uptake in 3T3-L1 adipocytes with an IC(50) of 20 microM. We conclude that apart from acting as a general tyrosine kinase inhibitor, genistein also affects the function of other proteins such as the GLUT4 transporter. These data suggest that caution must be applied when interpreting data on the involvement of tyrosine kinase activity in glucose uptake in 3T3-L1 adipocytes.

Osteoprotegerin in human milk: a potential role in the regulation of bone metabolism and immune development.

Osteoprotegerin (OPG) is a member of the tumor necrosis factor superfamily. It is a soluble "decoy" receptor for tumor necrosis factor-related apoptosis-inducing ligand and ligand of the receptor activator of NF-kappaB. As such, OPG inhibits osteoclast activity and regulates the immune system. Human milk is a complex biologic fluid that supplies nutritional and protective factors to the breast-fed infant. In the present study, human milk samples at various times postpartum were assessed for the presence of OPG. Using biochemical as well as immunologic and biologic techniques we showed that human milk contains OPG at a level that is 1000-fold higher than that found in normal human serum. We observed that human breast milk cells and the human mammary epithelial cell line MCF-7 express OPG, indicating that both cell types are possible sources of milk OPG in vivo. In vitro studies demonstrated that milk OPG is biologically active and suggested that it may contribute to the antiresorptive activity of milk on bone, as well as tumor necrosis factor-related apoptosis-inducing ligand-induced inhibition of T cell proliferation. OPG-like activity was also observed in bovine colostrum and milk. Furthermore, we were able to detect human OPG in the sera of rats gavaged with human milk. We discuss the relevance of our findings for the breast-fed infant and for the prevention of immune and bone disorders.

Protein expression and secretion in the yeast Yarrowia lipolytica.

Strains and vectors for protein expression and secretion have been developed in the yeast Yarrowia lipolytica. Host strains were constructed with non-reverting auxotrophic markers, deletions of protease-encoding genes, and carrying a docking platform. To drive transcription, either the synthetic hp4d or the inducible POX2 promoter were used. Protein secretion is either directed by the targeting sequence of the alkaline extracellular protease or the extracellular lipase (LIP2p) signal sequence. We describe a set of vectors based on these promoters, targeting sequences and two URA3 alleles as selection markers. The wild-type URA3 allele, ura3d1, was used for single-copy integration and a mutant URA3 allele, ura3d4, was used to select for multi-copy integration into the genome. These vectors were used to express the Y. lipolytica extracellular lipase LIP2p and the Aspergillus oryzae leucine amino peptidase II. Lipase production under the control of the hp4d promoter by a strain containing a single copy reached 1000 U ml(-1) in shake flasks, while a strain containing multiple integrations reached 2000 U ml(-1) in shake flasks, 11500 U ml(-1) in batch and 90500 U ml(-1) in fed batch. Leucine amino peptidase production under the control of the hp4d promoter reached 320 mU ml(-1) in batch with a mono-copy lapA integrant and 28000 mU ml(-1) in fed batch with a multi-copy transformant.