Language mapping in healthy volunteers and brain tumor patients with a novel navigated TMS system: evidence of tumor-induced plasticity.

OBJECTIVE: This article explores the feasibility of a novel repetitive navigated transcranial magnetic stimulation (rnTMS) system and compares language mapping results obtained by rnTMS in healthy volunteers and brain tumor patients. METHODS: Fifteen right-handed healthy volunteers and 50 right-handed consecutive patients with left-sided gliomas were examined with a picture-naming task combined with time-locked rnTMS (5-10 Hz and 80-120% resting motor threshold) applied over both hemispheres. Induced errors were classified into four psycholinguistic types and assigned to their respective cortical areas according to the coil position during stimulation. RESULTS: In healthy volunteers, language disturbances were almost exclusively induced in the left hemisphere. In patients errors were more frequent and induced at a comparative rate over both hemispheres. Predominantly dysarthric errors were induced in volunteers, whereas semantic errors were most frequent in the patient group. CONCLUSION: The right hemisphere's increased sensitivity to rnTMS suggests reorganization in language representation in brain tumor patients. SIGNIFICANCE: rnTMS is a novel technology for exploring cortical language representation. This study proves the feasibility and safety of rnTMS in patients with brain tumor.

A new approach for corticospinal tract reconstruction based on navigated transcranial stimulation and standardized fractional anisotropy values.

PURPOSE: To establish a novel approach for fiber tracking based on navigated transcranial magnetic stimulation (nTMS) mapping of the primary motor cortex and to propose a new algorithm for determination of an individualized fractional anisotropy value for reliable and objective fiber tracking. METHODS: 50 patients (22 females, 28 males, median age 58 years (20-80)) with brain tumors compromising the primary motor cortex and the corticospinal tract underwent preoperative MR imaging and nTMS mapping. Stimulation spots evoking muscle potentials (MEP) closest to the tumor were imported into the fiber tracking software and set as seed points for tractography. Next the individual FA threshold, i.e. the highest FA value leading to visualization of tracts at a predefined minimum fiber length of 110 mm, was determined. Fiber tracking was then performed at a fractional anisotropy value of 75% and 50% of the individual FA threshold. In addition, fiber tracking according to the conventional knowledge-based approach was performed. Results of tractography of either method were presented to the surgeon for preoperative planning and integrated into the navigation system and its impact was rated using a questionnaire. RESULTS: Mapping of the motor cortex was successful in all patients. A fractional anisotropy threshold for corticospinal tract reconstruction could be obtained in every case. TMS-based results changed or modified surgical strategy in 23 of 50 patients (46%), whereas knowledge-based results would have changed surgical strategy in 11 of 50 patients (22%). Tractography results facilitated intraoperative orientation and electrical stimulation in 28 of 50 (56%) patients. Tracking at 75% of the individual FA thresholds was considered most beneficial by the respective surgeons. CONCLUSIONS: Fiber tracking based on nTMS by the proposed standardized algorithm represents an objective visualization method based on functional data and provides a valuable instrument for preoperative planning and intraoperative orientation and monitoring.

Effects of new insulin analogues HMR1964 (insulin glulisine) and HMR1423 on insulin receptors.

AIMS/HYPOTHESIS: New insulin analogues have been created by amino-acid exchange to provide an improved pharmacokinetic profile. However, safety issues have been raised regarding their use, as amino-acid exchange of insulin may induce altered metabolic and mitogenic effects. For example, the insulin analogue Asp(B10) causes breast cancer in rodents. The aim of this study was to compare two new insulin analogues HMR1964 (Lys[B3],Glu[B29]) (insulin glulisine) and HMR1423 (Gly[A21],His[B31],His[B32]) with regular insulin and the mitogenic analogue Asp(B10). MATERIALS AND METHODS: We analysed insulin receptor binding characteristics and dissociation kinetics, as well as insulin-induced receptor auto- and dephosphorylation kinetics, in rat-1 fibroblasts overexpressing the human insulin receptor isoform B. Mitogenic activity was tested in the non-malignant cell line MCF10. RESULTS: Regular insulin, HMR1964 and HMR1423 showed no significant differences in receptor association, dissociation and receptor binding affinity, while Asp(B10) displayed markedly increased insulin receptor affinity. All of the analogues induced rapid insulin receptor autophosphorylation, reaching a maximum 10 min after stimulation (10(-9) mmol/l insulin). In contrast, Asp(B10) induced a prolonged phosphorylation and dephosphorylation state of the 95 kDa insulin receptor beta-subunit. With respect to [3H]thymidine incorporation, the new analogues had similar (HMR1423) or even lower (HMR1964) effects than regular insulin in the mammary epithelial cell line MCF10, while Asp(B10) showed increased [3H]thymidine incorporation. CONCLUSIONS/INTERPRETATION: HMR1964 and HMR1423 displayed the same association, dissociation and insulin receptor affinity kinetics as regular insulin, and might therefore be useful for the treatment of diabetes.

Insulin inhibits leptin receptor signalling in HEK293 cells at the level of janus kinase-2: a potential mechanism for hyperinsulinaemia-associated leptin resistance.

AIMS/HYPOTHESIS: Leptin resistance in obese humans seems to be predominantly caused by signalling abnormalities at the post receptor level. Leptin resistance in obese individuals is frequently associated with insulin resistance and pronounced hyperinsulinaemia indicating a negative crosstalk of the insulin and leptin signalling chain. METHODS: This hypothesis was tested using a cell model of peripheral leptin signalling, i. e. insulin-secreting cell lines (RINr1046-38). Mechanisms for a crosstalk between the insulin and leptin signalling pathway were also studied in rat-1 and HEK293 cells overexpressing elements of the insulin and leptin signalling chain. RESULTS: The effects of leptin on insulin secretion are completely cancelled by a 4-h preincubation with 1 nmol/l insulin, supporting the hypothesis of a negative crosstalk of insulin and leptin signalling. We investigated the potential molecular mechanisms in more detail in HEK293 cells and Rat-1 fibroblasts that overexpressed proteins of the insulin and leptin signalling chain. Leptin (60 ng/ml) stimulated autophosphorylation of JAK-2 in HEK 293 cells. This leptin effect could be inhibited by simultaneous treatment of cells with insulin. Furthermore, overexpression of the insulin receptor in HEK 293 cells clearly reduced JAK-2 phosphorylation and led further downstream to a diminished phosphatidylinositol 3-kinase activity. The inhibitory effect of the insulin signal could be partially prevented by transfection of the cells with an inactive mutant of the tyrosine phosphatase SHP-1. CONCLUSION/INTERPRETATION: In summary, our data suggest that the insulin receptor signalling pathway interferes with leptin signalling at the level of JAK-2. Inhibition of JAK-2 phosphorylation might occur through SHP-1-dependent pathways, indicating that hyperinsulinaemia contributes to the pathogenesis of leptin resistance.

Inhibition of Ret oncogene activity by the protein tyrosine phosphatase SHP1.

Germline mutations in the Ret protooncogene give rise to the inherited endocrine cancer syndromes MEN types 2A and 2B and familiar medullary thyroid carcinoma. Although it is well accepted that the constitutive active tyrosine kinase of Ret oncogenes ultimately leads to malignant transformation, it is not clear whether a decrease in the autophosphorylation of oncogenic Ret forms can affect the mitogenic and transforming activities of Ret. Potential modulators of the tyrosine kinase activity of Ret could be tyrosine phosphatases that are expressed in human thyroid tissue. Therefore, we investigated the impact of the tyrosine phosphatases SHP1 and SHP2 on the intrinsic tyrosine kinase activity and oncogenic potency of Ret with a 9-bp duplication in the cysteine-rich domain (codons 634-636), which was described in a patient with MEN type 2A recently. SHP1 and SHP2 were stably overexpressed in NIH3T3 fibroblasts together with Ret-9bp. Coexpression of SHP1 with Ret-9bp reduced the autophosphorylation of Ret-9bp by 19 +/- 7% (P = 0.01, n = 4), whereas no effect was seen with SHP2. Furthermore, Ret-9bp could be coimmunoprecipitated with SHP1 but not with SHP2 antibodies. Suppression of the Ret-9bp tyrosine kinase activity by SHP1 caused a decrease in activation of Erk2 (extracellular signal-regulated kinase) and abolished PKB/Akt (protein kinase B) phosphorylation. In addition, diminished Ret-9bp autophosphorylation led to reduced phosphorylation of the transcription factor jun-D. Finally, the inhibitory effect on Ret-9bp signaling resulted in a 40-60% reduction of [(3)H]thymidine incorporation and in reduced ability of NIH3T3 cells to form colonies in soft agar. In conclusion, the data suggest that SHP1 caused a moderate reduction of Ret autophosphorylation, which led to a strong suppression of the Ret oncogene activity.

Ret oncogene signal transduction via a IRS-2/PI 3-kinase/PKB and a SHC/Grb-2 dependent pathway: possible implication for transforming activity in NIH3T3 cells.

Multiple endocrine neoplasia 2A (MEN 2A) is an inherited disease caused by mutations of the Ret proto-oncogene. Although many different Ret mutations have been described, little is known about the signaling pathways triggered by the Ret oncogene. In this study, we have determined the signaling properties of a Ret-9bp duplication encoding amino acids 634-636, which was recently identified in a patient with all clinical features of the MEN 2A syndrome. The Ret-9bp duplication leads to constitutive activation of the Ret tyrosine kinase. Furthermore, Ret-9bp increased mitogenic and transforming activity demonstrated by thymidine incorporation as well as colony formation in soft agar. Studying intracellular signaling pathways, which may be involved in malignant transformation of Ret-9bp expressing NIH3T3 cells, we could demonstrate Ret-9bp dependent phosphorylation of insulin receptor substrate-2 (IRS-2) with consecutive activation of phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (PKB/AKT). Moreover, Ret-9bp induces phosphorylation of SHC resulting in growth factor receptor binding protein-2 (Grb-2) binding and activation of the mitogen activating protein (MAP) kinase pathway. In addition to these postreceptor cytoplasmic signaling events, we have studied nuclear signal by Ret-9bp and found activation of c-jun and jun-D, two members of the jun/AP-1 family of transcription factors. In summary, an oncogenic 9bp duplication of Ret causes Ret dimer formation and ligand independent activation of the tyrosine kinase. Besides the signaling steps leading to MAPK activation, we could demonstrate that Ret-9bp induced constitutive activation of a signaling pathway involving IRS-2, PI 3-kinase and PKB/AKT which could transduce the oncogenic Ret signal to increased gene transcription via activation of the jun/AP-1 transcription factor family.

Serine residues 1177/78/82 of the insulin receptor are required for substrate phosphorylation but not autophosphorylation.

Serine residues of the human insulin receptor (HIR) may be phosphorylated and negatively regulate the insulin signal. We studied the impact of 16 serine residues in HIR by mutation to alanine and co-overexpression in human embryonic kidney (HEK) 293 cells together with the docking proteins insulin receptor substrate (IRS)-1, IRS-2, or (SHC) Src homologous and collagen-like. As a control, IRS-1 was also cotransfected with an HIR with a juxtamembrane deletion (HIR delta JM) and therefore not containing the domain required for interaction with IRS-1. Coexpression of HIR with IRS-1, IRS-2, and SHC strongly enhanced tyrosine phosphorylation of these proteins. A similar increase in tyrosine phosphorylation was observed in cells overexpressing IRS-1, IRS-2, or SHC together with all HIR mutants except HIR delta JM and a mutant carrying exchanges of serines 1177, 1178, and 1182 to alanine (HIR1177/78/82), although this mutant showed normal autophosphorylation. Analysis of total cell lysates with anti-phosphotyrosine antibodies showed that in addition to the overexpressed substrates, other cellular proteins displayed reduced levels of tyrosine phosphorylation in these cells. To study consequences for phosphatidylinositol 3-kinase (PI 3-kinase) activation, we established stable NIH3T3 fibroblast cell lines overexpressing wild-type HIR, HIR1177/78/82, and other HIR mutants as the control. Again, HIR1177/78/82 showed normal autophosphorylation but showed a clear decrease in tyrosine phosphorylation of endogenous IRS-1 and activation of PI 3-kinase. This decrease in kinase activity also occurred in an in vitro kinase assay towards recombinant IRS-1. Finally, we performed a separation of the phosphopeptides by high-performance liquid chromatography and could not detect any differences in the profiles of HIR and HIR1177/78/82. In conclusion, we have defined a region in HIR that is important for substrate phosphorylation but not autophosphorylation. Therefore, this mutant may provide new insights into the mechanism of kinase activation and substrate phosphorylation.

Serine residues 994 and 1023/25 are important for insulin receptor kinase inhibition by protein kinase C isoforms beta2 and theta.

AIMS/HYPOTHESIS: Inhibition of the signalling function of the human insulin receptor (HIR) is one of the principle mechanisms which induce cellular insulin resistance. It is speculated that serine residues in the insulin receptor beta-subunit are involved in receptor inhibition either as inhibitory phosphorylation sites or as part of receptor domains which bind inhibitory proteins or tyrosine phosphatases. As reported earlier we prepared 16 serine to alanine point mutations of the HIR and found that serine to alanine mutants HIR-994 and HIR-1023/25 showed increased tyrosine autophosphorylation when expressed in human embryonic kidney (HEK) 293 cells. In this study we examined whether these mutant receptors have a different susceptibility to inhibition by serine kinases or an altered tyrosine kinase activity. METHODS: Tyrosine kinase assay and transfection studies. RESULTS: In an in vitro kinase assay using IRS-1 as a substrate we could detect a higher intrinsic tyrosine kinase activity of both receptor constructs. Additionally, a higher capacity to phosphorylate the adapter protein Shc in intact cells was seen. To test the inhibition by serine kinases, the receptor constructs were expressed in HEK 293 cells together with IRS-1 and protein kinase C isoforms beta2 and theta. Phorbol ester stimulation of these cells reduced wild-type receptor autophosphorylation to 58 % or 55 % of the insulin simulated state, respectively. This inhibitory effect was not observed with HIR-994 and HIR-1023/25, although all other tested HIR mutants showed similar inhibition induced by protein kinase C. CONCLUSION/INTERPRETATION: The data suggest that the HIR-domain which contains the serine residues 994 and 1023/25 is important for the inhibitory effect of protein kinase C isoforms beta2 and theta on insulin receptor autophosphorylation.

The inhibitory effect of 2-deoxyglucose on insulin receptor autophosphorylation does not depend on known serine phosphorylation sites or other conserved serine residues of the receptor beta-subunit.

Hyperglycemia induces insulin resistance in diabetic patients. It is known that supraphysiological levels of D-glucose or 2-deoxyglucose inhibit the insulin receptor and it is speculated that this effect is mediated by serine phosphorylation of the insulin receptor beta-subunit and other proteins of the insulin signaling chain. To test this hypothesis we prepared point mutations of the human insulin receptor where serine was exchanged to alanine at 16 different positions, either at known phosphorylation sites or at positions which are conserved in different tyrosine kinase receptors. These receptor constructs were expressed in HEK 293 cells and the effect of 2-deoxyglucose (25 mM) on insulin (100 nM) induced receptor autophosphorylation was studied. 2-Deoxyglucose consistently inhibits insulin stimulated autophosphorylation of all constructs to the same degree as observed in wild-type human insulin receptor. The data suggest that none of the chosen serine positions are involved in 2-deoxyglucose induced receptor inhibition.

Impact of mutations at different serine residues on the tyrosine kinase activity of the insulin receptor.

Insulin binding to its receptor activates a cascade of signaling events which are initiated by tyrosine autophosphorylation of the receptor and activation of the tyrosine kinase activity towards the insulin receptor substrates. In addition to phosphorylation at tyrosine residues a serine phosphorylation of the insulin receptor is observed. Neither the functional significance of serine phosphorylation of the receptor nor the location of relevant regulatory sites has been determined exactly so far. We studied potential functions of serine residues in human insulin receptor (HIR) with respect to its ability to undergo insulin stimulated autophosphorylation. Using site directed mutagenesis of HIR we exchanged serine to alanine at 13 different positions in the HIR beta-subunit. Sites were chosen according to the criteria of known serine phosphorylation sites (1023/25, 1293/94, 1308/09), conserved positions in hIR, hIGF-1 receptor, hIRR, and dIR (962, 994, 1037, 1055, 1074/78, 1168, 1177/78/82, 1202, 1263, 1267). All HIR mutants were expressed in HEK 293 cells and basal and insulin stimulated autophosphorylation were determined. We found that the exchange of serine to alanine at position 994 and at position 1023/25 increased insulin stimulated receptor autophosphorylation significantly (147% +/- 12% and 129% +/- 6% of control, p < 0.01, n = 7), while all other exchanges did not significantly alter insulin stimulated HIR autophosphorylation. The data suggest that the serine residues at position 994 as well as 1023/25 might be part of inhibitory domains of the insulin receptor.

A 973 valine to methionine mutation of the human insulin receptor: interaction with insulin-receptor substrate-1 and Shc in HEK 293 cells.

A population-based study in the Netherlands has recently demonstrated that a mutation of the human insulin receptor (HIR-973 valine to methionine) is associated with hyperglycaemia and an increased prevalence of non-insulin-dependent diabetes mellitus (NIDDM). The aim of the present study was to assess whether this mutation leads to a functional alteration of the insulin receptor. We prepared the HIR-973 mutant by in vitro mutagenesis. This mutant was transiently overexpressed in HEK 293 cells either alone or together with insulin-receptor substrate-1 (IRS-1) or Shc. Insulin stimulated autophosphorylation, phosphorylation of the substrates IRS-1 and Shc as well as activation of phosphatidylinositol-3 (PI3)-kinase were studied. Autophosphorylation of HIR-973 and its susceptibility to hyperglycaemia induced inhibition was not different from HIR-wt. Human insulin receptor with a juxtamembrane deletion HIR-deltaJM which is known to impair HIR/IRS-1 interaction was used as control. While the HIR-deltaJM induces a reduced IRS-1 phosphorylation HIR-973 showed even a slightly increased ability to phosphorylate IRS-1 (n = 7, 115% of control, p < 0.01). Shc phosphorylation was only mediated by HIR-wt and HIR-973 but not by HIR-deltaJM. Again a tendency to higher phosphorylation of Shc was seen with HIR-973 (n = 7, 109% of control, NS). When PI3-kinase activity was measured in IRS-1 precipitates similar activity was found for HIR-wt and HIR-973 whereas PI3-kinase stimulation was reduced with HIR-deltaJM. In summary, the data suggest that HIR-973 does not impair the first steps of the insulin signalling cascade. It is therefore unlikely that this mutation may cause cellular insulin resistance. The close vicinity of this mutation to insulin receptor domains which are involved in IRS-1 and Shc binding may, however, alter the interaction of the insulin receptor with these substrates. This could explain the slightly increased insulin effect on tyrosine phosphorylation of these docking proteins. These characteristics of HIR-973 might have a compensatory function of impaired signal transduction further downstream of the signalling chain in this specific subgroup of NIDDM patients.