Kristina S. Kovacina

Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase.

In the present study, insulin is shown to rapidly stimulate by 8‐ to 12‐fold the enzymatic activity of RAC‐PK alpha, a pleckstrin homology domain containing ser/thr kinase. In contrast, activation of protein kinase C by phorbol esters had almost no effect on the enzymatic activity of RAC‐PK alpha. Insulin activation was accompanied by a shift in molecular weight of the RAC‐PK alpha protein, and the activated kinase was deactivated by treatment with a phosphatase, indicating that insulin activated the enzyme by stimulating its phosphorylation. This insulin‐induced shift in RAC‐PK was also observed in primary rat epididymal adipocytes, as well as in a muscle cell line called C2C12 cells. The insulin‐stimulated increase in RAC‐PK alpha activity was inhibited by wortmannin (an inhibitor of phosphatidylinositol 3‐kinase) in a dose‐dependent manner with a half‐maximal inhibition of 10 nM, but not by 20 ng/ml of rapamycin. Activation of RAC‐PK alpha activity was also observed in a variant RAC lacking the pleckstrin homology domain. These results indicate that RAC‐PK alpha activity can be regulated by the insulin receptor. RAC‐PK alpha may therefore play a general role in intracellular signaling mediated by receptor tyrosine kinases.

Reduced Hippocampal Insulin-Degrading Enzyme in Late-Onset Alzheimer's Disease Is Associated with the Apolipoprotein E-ε4 Allele

Abeta is the major component of amyloid plaques characterizing Alzheimers disease (AD). Abeta accumulation can be affected by numerous factors including increased rates of production and/or impaired clearance. Insulin-degrading enzyme (IDE) has been implicated as a candidate enzyme responsible for the degradation and clearance of Abeta in the brain. We have previously shown that AD patients exhibit abnormalities in insulin metabolism that are associated with apoliprotein E (APOE) status. The possible association of IDE with AD, as well as the link between APOE status and insulin metabolism, led us to examine the expression of IDE in AD. We report that hippocampal IDE protein is reduced by approximately 50% in epsilon4+ AD patients compared to epsilon4- patients and controls. The allele-specific decrease of IDE in epsilon4+ AD patients is not associated with neuronal loss since neuron-specific enolase levels were comparable between the AD groups, regardless of APOE status. Hippocampal IDE mRNA levels were also reduced in AD patients with the epsilon4 allele compared to AD and normal subjects without the epsilon4 allele. These findings show that reduced IDE expression is associated with a significant risk factor for AD and suggest that IDE may interact with APOE status to affect Abeta metabolism.

Interactions of recombinant and platelet transforming growth factor-β1 precursor with the insulin-like growth factor II/mannose 6-phosphate receptor

Recombinant transforming growth factor (TGF)-beta 1 precursor was recently found to contain mannose 6-phosphate (Purchio et al., 1988, J. Biol. Chem. 263, 14211-14215). In the present study, recombinant TGF-beta 1 precursor was shown to bind to the insulin-like growth factor (IGF)-II/mannose 6-phosphate (man6P) receptor on the plasma membrane of cells since: 1) Insulin, which induces an increase in cell surface IGF-II/man6P receptors on adipocytes, caused a 2.7-fold increase in TGF-beta 1 precursor binding to adipocytes; 2) Chinese hamster ovary cells selected for overexpression of the IGF-II/man6P receptor exhibited an increased binding of TGF-beta 1 precursor in comparison to the parental cells; and 3) the binding of 125I-TGF-beta 1 precursor to these transfected cells and adipocytes was largely inhibited by man6P. After 15 minutes at 37 degrees C, 75% of the recombinant TGF-beta 1 precursor was found to be internalized in the transfected cells. Additional studies with latent TGF-beta 1 isolated from platelets indicated that this material could also bind to the isolated IGF-II/man6P receptor.

Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation

cGMP‐inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin‐like growth factor‐1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP‐dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell‐free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP‐dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr‐Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin‐dependent maturation of Xenopus oocytes and rescues the phenotype of pde3−/− mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290–292 to alanine in both Xenopus and mouse. Microinjection of myr‐Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt‐mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.

Protein kinase B/Akt is essential for the insulin- but not progesterone-stimulated resumption of meiosis in Xenopus oocytes.

In the present study, we have characterized the Xenopus Akt expressed in oocytes from the African clawed frog Xenopus laevis and tested whether its activity is required for the insulin- and progesterone-stimulated resumption of meiosis. A cDNA encoding the Xenopus Akt was isolated and sequenced, and its expression in the Xenopus oocyte was confirmed by reverse transcription PCR and Northern blotting. Using phosphospecific antibodies and enzyme assays, a large and rapid activation of the Xenopus Akt was observed upon insulin stimulation of the oocytes. In contrast, progesterone caused a modest activation of this kinase with a slower time course. To test whether the activation of Akt was required in the stimulation of the resumption of meiosis, we have utilized two independent approaches: a functional dominant negative Akt mutant and an inhibitory monoclonal antibody. Both the mutant Akt, as well as the inhibitory monoclonal antibody, completely blocked the insulin-stimulated resumption of meiosis. In contrast, both treatments only partially inhibited (by approx. 30%) the progesterone-stimulated resumption of meiosis when submaximal doses of this hormone were utilized. These data demonstrate a crucial role for Akt in the insulin-stimulated cell cycle progression of Xenopus oocytes, whereas Akt may have an ancillary function in progesterone signalling.

PRAS40 plays a pivotal role in protecting against stroke by linking the Akt and mTOR pathways

The proline-rich Akt substrate of 40kDa (PRAS40) protein is not only a substrate of the protein kinase Akt but also a component of the mTOR complex 1 (mTORC1), thus it links the Akt and the mTOR pathways. We investigated the potential protective role of PRAS40 in cerebral ischemia and its underlying mechanisms by using rats with lentiviral over-expression of PRAS40 and mice with PRAS40 gene knockout (PRAS40 KO). Our results show that gene transfer of PRAS40 reduced infarction size in rats by promoting phosphorylation of Akt, FKHR (FOXO1), PRAS40, and mTOR. In contrast, PRAS40 KO increased infarction size. Although the PRAS40 KO under normal condition did not alter baseline levels of phosphorylated proteins in the Akt and mTOR pathways, PRAS40 KO that underwent stroke exhibited reduced protein levels of p-S6K and p-S6 in the mTOR pathway but not p-Akt, or p-PTEN in the Akt pathway. Furthermore, co-immunoprecipitation suggests that there were less interactive effects between Akt and mTOR in the PRAS40 KO. In conclusion, PRAS40 appears to reduce brain injury by converting cell signaling from Akt to mTOR.

The Insulin Receptor Substrate (IRS)-1 Pleckstrin Homology Domain Functions in Downstream Signaling*

The pleckstrin homology (PH) domain of the insulin receptor substrate-1 (IRS-1) plays a role in directing this molecule to the insulin receptor, thereby regulating its tyrosine phosphorylation. In this work, the role of the PH domain in subsequent signaling was studied by constructing constitutively active forms of IRS-1 in which the inter-SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase was fused to portions of the IRS-1 molecule. Chimeric molecules containing the PH domain were found to activate the downstream response of stimulating the Ser/Thr kinase Akt. A chimera containing point mutations in the PH domain that abolished the ability of this domain to bind phosphatidylinositol 4,5-bisphosphate prevented these molecules from activating Akt. These mutations also decreased by about 70% the amount of the constructs present in a particulate fraction of the cells. These results indicate that the PH domain of IRS-1, in addition to directing this protein to the receptor for tyrosine phosphorylation, functions in the ability of this molecule to stimulate subsequent responses. Thus, compromising the function of the PH domain, e.g. in insulin-resistant states, could decrease both the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor and to link to subsequent downstream targets.

On the mechanism for neomycin reversal of wortmannin inhibition of insulin-stimulation of glucose uptake

Although a number of studies and approaches have indicated that activation of the Ser/Thr kinase called Akt/protein kinase B is critical for the insulin-stimulated increase of glucose uptake in adipocytes, other studies have indicated that this enzyme may play an ancillary role. For example, a recent study indicated that neomycin would allow insulin-stimulated Glut4 translocation and glucose transport in the presence of the phosphatidylinositol (PI) 3-kinase inhibitor, wortmannin, a known inhibitor of Akt activation (James, D. J., Salaün, C., Brandie, F. M., Connell, J. M. C., and Chamberlain, L. H. (2004) J. Biol. Chem. 279, 20567–20570). To better understand this observation, we examined a number of downstream targets of Akt. As previously reported, treatment of 3T3-L1 adipocytes with neomycin prevented the wortmannin inhibition of insulin-stimulated glucose transport. However, in the presence of neomycin, wortmannin did not inhibit the insulin-stimulated phosphorylation of several downstream targets of Akt including a proline-rich Akt substrate of 40 kDa, ribosomal protein S6, and glycogen synthase kinase-3. In addition, neomycin did not prevent the ability of a structurally unrelated PI 3-kinase inhibitor, LY294002, to inhibit the insulin-stimulated activation of glucose uptake. Moreover, neomycin reversed the inhibitory effect of wortmannin but not LY294002 on insulin stimulation of Akt kinase activity. Finally, neomycin was found to inactivate in vitro the PI 3-kinase inhibitory actions of wortmannin but not LY294002. These results indicate that the effects of neomycin in adipocytes are not mediated via its ability to sequester phosphatidylinositol 4,5-bisphosphate but are instead caused by the ability of neomycin to inactivate wortmannin.

The Insulin Receptor Family

The insulin receptor family in mammals includes the receptors for insulin, insulin-like growth factor I (IGF-I), and the insulin receptor-related receptor (IRR), a receptor whose sequence is homologous to the sequences of the other two receptors but whose ligand is unknown (Fig. 1) (1). Another potential receptor in this family is the receptor for relaxin, a hormone whose structure is related to that of insulin (2). In addition, other members of this receptor family could exist for IGF-I and the highly related IGF-II. Although the cDNA and gene for one receptor which binds IGF-I with high affinity has been isolated (3), several pieces of data suggest that there may be other related IGF receptors. For example, although the receptor expressed from this cDNA had high affinity for IGF-I and IGF-II but almost 1000-fold weaker affinity for insulin (4), several reports in the literature have indicated that there are IGF receptors with a much higher affinity for insulin or a much weaker affinity for IGF-II (5, 6). Some of these studies may have been affected by the presence of hybrid insulin-IGF-I receptors (7). In addition, expression of the apparently same cDNA for the IGF-I receptor in another cell type led to the formation of a receptor which had high affinity for IGF-I and weak affinity for IGF-II when binding studies were performed on whole cells but which bound IGF-I and II almost equally when binding studies were performed in cell lysates (8). In addition, several monoclonal antibodies to the IGF-I receptor were found to stimulate an increase in the affinity of the IGF-I receptor for insulin almost to the level of the insulin receptor (9). These results suggest that the same IGF receptor can bind the IGFs and insulin with different relative affinities depending on the environment of the receptor in the cell membrane. Thus, it is not clear at the present time whether another IGF receptor exists or whether these different binding data can be explained by the present IGF-I receptor interacting with other molecules in different cell backgrounds.