Haptic interface for robot-assisted ophthalmic surgery.

Vitreo-retinal surgery is challenging, as delicate structures have to be manipulated. Eliminating tremor caused by human motions when doing micromanipulation can therefore improve the outcome of such an intervention. An eye surgery robot has been built to overcome this problem. The contribution of this paper is the design of a telemanipulation setup for the robotic system. A telemanipulation setup using a haptic device featuring force feedback as a user interface for controlling a hybrid parallel-serial micromanipulator is designed and developed. The position error control scheme is chosen and different control modes are provided. The output forces of the haptic device are analyzed. The system allows the surgeon to perform precise and comfortable micromanipulation. Nevertheless a way to provide more meaningful force feedback still has to be found.

Genetics and the clinical approach to paragangliomas.

This study analyses new information on gene mutations in paragangliomas and puts them into a clinical context. A suspicion of malignancy is critical to determine the workup and surgical approach in adrenal (A-PGL) and extra-adrenal (E-PGL) paragangliomas (PGLs). Malignancy rates vary with location, family history, and gene tests results. Currently there is no algorithm incorporating the above information for clinical use. A sum of 1,821 articles were retrieved from PubMed using the search terms "paraganglioma genetics". Thirty-seven articles were selected of which 9 were analyzed. It was found that 599/2,487 (24%) patients affected with paragangliomas had a germline mutation. Of these 30.2% were mutations in SDHB, 25% VHL, 19.4% RET, 18.4% SDHD, 5.0% NF1, and 2.0% SDHC genes. A family history was positive in 18.1-64.3% of patients. Adrenal PGLs accounted for 55.1% in mutation (+) and 81.0% in mutation (-) patients (RR 1.2, p < 0.0001). Bilateral A-PGLs accounted for 56.4% in mutation (+) and 3.2% in mutation (-) patients (RR 8.7, p < 0.0001). E-PGL were found in 33.6% of mut+ and 17.3% of mut- (RR 1.7, p < 0.0001). In mutation (+) patients PGLs malignancy varied with location, adrenal (6.4%) thoraco-abdominal E-PGL (38%), H & N E-PGL (10%). Malignancy rates were 8.2% in mutation (-) and lower in mutation (+) PGLs except for SDHB 36.5% and SDHC 8.3%. Exclusion of a mutation lowered the probability of malignancy significantly in E-PGL (RR 0.03 (95% CI 0.1-0.6); p < 0.001). Mutation analysis provides valuable preoperative information to assess the risk of malignancy in A-PG and E-PGLs and should be considered in the work up of all E-PGL lesions.

Oncologic resection achieving r0 margins improves disease-free survival in parathyroid cancer.

BACKGROUND: Parathyroid cancer has a poor mid-term prognosis, often because of local recurrence, observed in half of all patients. Modern diagnostic workup increasingly enables a preoperative diagnosis of parathyroid cancer. There is limited evidence that more comprehensive oncologic surgery can reduce the risk of local recurrence. This study aims to identify the best specific surgical approach in parathyroid cancer. METHODS: This observational cohort study comprises 19 consecutive patients who had undergone oncologic or nononcologic resection for parathyroid cancer. Baseline parameters were compared by using univariate analysis; outcomes were assessed by χ (2) testing and Kaplan-Meier statistics. RESULTS: Fifteen of 19 patients were primarily operated on in our tertiary center between 1996 and 2013, and four were referred for follow-up because of their cancer diagnosis. Patient cohorts defined by histologic R-status were comparable for established risk factors: sex, calcium levels, low-risk/high-risk status, and presence of vascular invasion. Oncologic resections were performed in 13 of 15 patients primarily treated in the center and 0 of 4 treated elsewhere (χ (2) = 5.6; p < 0.01). R0 margins were achieved in 11 of 13 (85 %) undergoing oncologic resection and 1 of 6 (17 %) undergoing local excision (χ (2) = 8.1; p < 0.01). R0 margins and primary oncologic resection were associated with higher disease-free survival rates (χ (2) = 7.9; p = 0.005 and χ (2) = 4.7; p = 0.03, respectively). Revision surgery achieved R0 margins in only 2 of 4 (50 %) of patients. CONCLUSIONS: In parathyroid cancer, a more comprehensive surgery (primary oncologic resection) provides significantly better outcomes than local excision as a result of reduction of R1 margins and locoregional recurrence.

The EuroNet paediatric hodgkin network - modern imaging data management for real time central review in multicentre trials.

Since 2007, children and adolescents with Hodgkin lymphomas are treated in the Europe-wide EuroNet-PHL trials. A real time central review process for stratification of the patients enhances quality control and efficient therapy management. This process includes reading of all cross-sectional-images. Since reference evaluation is time critical, a fast, easy to handle and safe data transfer is important. In addition, immediate and constant access to all the data has to be guaranteed in case of queries and for regulatory reasons. To meet the mentioned requirements the EuroNet Paediatric Hodgkin Data Network (funded by the European Union - Project Number: 2007108) was established between 2008 and 2011. A respective tailored data protection plan was formulated. The aim of this article is to describe the networks' mode of operation and the advantages for multi-centre trials that include centralized image review.

Gastric exocrine and endocrine cell morphology under prolonged acid inhibition therapy: results of a 5-year follow-up in the LOTUS trial.

BACKGROUND: Sustained acid inhibition with PPI stimulates gastrin secretion, exerting a proliferative drive on enterochromaffin-like cells (ECL cells) of the oxyntic mucosa. It may also accelerate development of gastric gland atrophy in Helicobacter pylori-infected individuals. AIMS: To evaluate gastric exocrine and endocrine cell changes in GERD patients randomised to laparoscopic antireflux surgery (LARS, n = 288) or long-term (5 years) esomeprazole (ESO) treatment (n = 266). METHODS: Antral and corpus biopsies were taken at endoscopy and serum gastrin and chromogranin A levels were assayed, at baseline and after 1, 3 and 5 years' therapy. RESULTS: Biopsies were available at each time point for 158 LARS patients and 180 ESO patients. In H. pylori-infected subjects, antral mucosal inflammation and activity improved significantly (P < 0.001) and stabilised after 3 years on esomeprazole while no change in inflammation was observed after LARS. Oxyntic mucosal inflammation and activity remained stable on esomeprazole but decreased slightly over time after LARS. Neither intestinal metaplasia nor atrophy developed in the oxyntic mucosa. ECL cell density increased significantly after ESO (P < 0.001), corresponding with an increase in circulating gastrin and chromogranin A. After LARS, there was a significant decrease in ECL cell density (P < 0.05), accompanied by a marginal decrease in gastrin and chromogranin. CONCLUSIONS: Antral gastritis improved in H. pylori-infected GERD patients after 5 years on esomeprazole, with little change in laparoscopic antireflux surgery patients, who acted as a control. Despite a continued proliferative drive on enterochromaffin-like cells during esomeprazole treatment, no dysplastic or neoplastic lesions were found and no safety concerns were raised. NCT 00251927.

Oral involvement in sarcoidosis: report of 12 cases.

AIM: To assess the clinical features, treatment and outcome of oral sarcoidosis and to determine whether oral involvement is associated with a particular clinical phenotype of sarcoidosis. DESIGN: Multicentric retrospective study. METHODS: Retrospective chart review. Each patient was matched with four controls. RESULTS: Twelve patients (9 women, 3 men) were identified. Their median age at sarcoidosis diagnosis was 38 years. Oral involvement was the first clinical evidence of sarcoidosis in seven cases and was a relapse symptom in five cases. Clinical presentations were nodules (n = 7) or ulcers (n = 5) and were mostly solitary. The tongue was the commonest site affected (n = 4), followed by lips (n = 3), oral mucosa (n = 2), palate (n = 2) and gingiva (n = 1). Patients with oral sarcoidosis were significantly younger and had more frequent lacrimal or salivary glands and upper airway tract clinical involvement than the controls; increased angiotensin-converting enzyme was less frequent in oral sarcoidosis. Multiple treatments of oral sarcoidosis were used: no treatment (n = 3), surgery (n = 2), corticosteroids (n = 7), hydroxychloroquine (n = 3), methotrexate (n = 2), doxycycline (n = 1). Methotrexate was efficient in one patient, hydroxychloroquine showed benefit in only 1 out of 3 patients. Three patients presented oral relapses. After a mean follow-up of 6 years, 10 patients experienced a complete (n = 7) or partial (n = 3) remission of oral sarcoidosis; stability was observed in the remaining two cases. CONCLUSION: Although oral manifestations of sarcoidosis are unusual, physicians should be aware that this specific localization is frequently the first manifestation of the disease. Treatment modalities range from observation in asymptomatic patients to immunosuppressants for severe involvement.

Ceruloplasmin expression in rat liver cells is attenuated by insulin: role of FoxO transcription factors.

The phosphoinositide 3'-kinase (PI3 K)/Akt pathway controls the activity of a number of proteins important in the regulation of apoptosis and cell proliferation. FoxO (forkhead box, class O) transcription factors, substrates of the Ser/Thr kinase Akt, control the expression of several target genes that are crucial to the defense against oxidative stress, the regulation of cell cycle, and apoptosis in mammalian cells. Here, expression of ceruloplasmin (CP), the major copper-containing protein in blood released by the liver, was investigated. We observed a significant downregulation of CP mRNA levels after insulin treatment in H4IIE rat hepatoma cells. The PI3K inhibitor wortmannin counteracted this insulin effect on CP mRNA levels, indicating that the PI3K/Akt cascade is involved in the regulation of CP expression. Stimulation of FoxO1 was induced in H4IIE rat hepatoma cells expressing a conditionally active FoxO1 construct, resulting in significant upregulation of CP mRNA levels. This upregulation was prevented in the presence of insulin. In parallel, mRNAs of established FoxO target genes were analyzed: like CP mRNA, selenoprotein P and glucose 6-phosphatase mRNAs were upregulated by FoxO1, which was prevented by insulin. The same effects of insulin on CP mRNA levels were detected in primary rat hepatocytes. Furthermore, CP release into cell culture media was analyzed with primary hepatocytes and found to be attenuated by insulin. In line with its insulin-mimetic effects on cultured cells, Cu (2+) imitated the effect of insulin on CP expression and caused a downregulation of CP mRNA levels in rat hepatoma cells.

Alcohol consumption and other psycho-social conditions as important factors in the development of diabetic foot ulcers.

AIMS: To characterize bio-psycho-social factors, particularly mental disorders and self-harm behaviour, associated with the development of diabetic foot ulcers. METHODS: Two groups of diabetic patients with and without foot ulcers (n=47 in each group) with similar sex, age and diabetes duration were assessed for mental disorders using the Composite International Diagnostic Interview. Self-harm behaviour, quality of life, depressive symptoms and self-compassion were rated using different standard questionnaires. RESULTS: Patients from the ulcer group visited their practitioners and/or psychotherapists less frequently in the last 12 months than patients in the control group 0 vs. 13%; P=0.026). The ulcer group patients had a history of increased alcohol consumption (43 vs. 19%; P=0.025), lower levels of education (8 vs. 10 grades; P=0.014) and income (1190 vs. 1535 €/month; P=0.039). Additionally, they were less likely to be diagnosed with anxiety disorders (11 vs. 32%; P=0.022). No significant differences in glycated haemoglobin, body mass index, smoking and direct self-harm behaviour were identified. CONCLUSIONS: Patients with foot ulcers tend to exhibit lower health-conscious behaviour, particularly higher lifetime alcohol consumption, lower utilization of medical services and less general anxiety. Practitioners should be aware of these behaviours, since early detection of diabetes patients at psycho-social risk and consecutive psychological intervention may be an effective preventive strategy in avoiding the development of foot ulcers.

Classification of canine malignant lymphomas according to the World Health Organization criteria.

A study was carried out to test the accuracy and consistency of veterinary pathologists, not specialists in hematopathology, in applying the World Health Organization (WHO) system of classification of canine lymphomas. This study represents an initiative of the ACVP Oncology Committee, and the classification has been endorsed by the World Small Animal Veterinary Association (WASVA). Tissue biopsies from cases of canine lymphoma were received from veterinary oncologists, and a study by pathologists given only signalment was carried out on 300 cases. Twenty pathologists reviewed these 300 cases with each required to choose a diagnosis from a list of 43 B and T cell lymphomas. Three of the 20 were hematopathologists who determined the consensus diagnosis for each case. The 17 who formed the test group were experienced but not specialists in hematopathology, and most were diplomates of the American or European Colleges of Veterinary Pathology. The overall accuracy of the 17 pathologists on the 300 cases was 83%. When the analysis was limited to the 6 most common diagnoses, containing 80% of all cases, accuracy rose to 87%. In a test of reproducibility enabled by reintroducing 5% of cases entered under a different identity, the overall agreement between the first and second diagnosis ranged from 40 to 87%. The statistical review included 43,000 data points for each of the 20 pathologists.

Regulation of glucose-6-phosphatase gene expression by insulin and metformin.

The biguanide derivative metformin is a potent anti-diabetic drug widely used in the treatment of type 2 diabetes mellitus. Its major effect on glucose metabolism consists in the inhibition of hepatic glucose production. Since the mechanisms of metformin action are only partially understood at the molecular level, we studied the regulation of the gene promoter activity of glucose-6-phosphatase (G6Pase), the central hepatic gluconeogenic enzyme, by this drug. We have found that both metformin and insulin inhibit the basal and dexamethasone/cAMP-stimulated G6Pase promoter activity in hepatoma cells. Since one of the pharmacological targets of metformin is AMP-activated protein kinase (AMPK) and activation of AMPK is known to inhibit hepatic glucose production by the suppression of G6Pase gene transcription, we studied the effect of AMPK in this context. Under nonstimulated conditions, the inhibitory effect of both insulin and metformin was partially counteracted to a similar extent by treatment with compound C, a specific inhibitor of AMPK. In contrast, under conditions of stimulation with dexamethasone and cAMP, treatment with compound C reversed the inhibitory effect of metformin on G6Pase promoter activity to a similar extent as compared to nonstimulated conditions, whereas the effect of insulin was almost resistant to treatment with the AMPK-antagonist. These data indicate a differential AMPK-dependent regulation of G6Pase gene expression by insulin and metformin under basal and dexamethasone/cAMP-stimulated conditions.

Molecular mechanisms of insulin resistance.

Currently, we observe an epidemic expansion of diabetes mellitus. In subjects with Type 2 diabetes the resistance of fat, muscle and liver to insulin is the central pathophysiological event in the development of this disease. Genetic and environmental factors play a major role in this process, although the precise pathogenesis of insulin resistance and Type 2 diabetes is still largely unknown. However, recent studies have contributed to a deeper understanding of the molecular mechanisms underlying this process. In this review we therefore summarize the current developments in understanding the pathophysiological process of insulin resistance and Type 2 diabetes. Among the many molecules involved in the intracellular processing of the signal provided by insulin, insulin receptor substrate (IRS)-2, the protein kinase B (PKB)-beta isoform and the forkhead transcription factor Foxo1a (FKHR) are of particular interest in this context as recent data have provided strong evidence that dysfunction of these proteins results in insulin resistance in-vivo. Furthermore, we have now increasing evidence that the adipose tissue not only produces free fatty acids that contribute to insulin resistance, but also acts as a relevant endocrine organ producing mediators (adipokines) that can modulate insulin signalling. The identification of the molecular pathophysiological mechanisms of insulin resistance and Type 2 diabetes is essential for the development of novel and more effective therapies to better treat our patients with insulin resistance and Type 2 diabetes.

Human adipocytes secrete mineralocorticoid-releasing factors.

Obesity has become an epidemic problem in western societies, contributing to metabolic diseases, hypertension, and cardiovascular disease. Overweight and obesity are frequently associated with increased plasma levels of aldosterone. Recent evidence suggests that human fat is a highly active endocrine tissue. Therefore, we tested the hypothesis that adipocyte secretory products directly stimulate adrenocortical aldosterone secretion. Secretory products from isolated human adipocytes strongly stimulated steroidogenesis in human adrenocortical cells (NCI-H295R) with a predominant effect on mineralocorticoid secretion. Aldosterone secretion increased 7-fold during 24 h of incubation. This stimulation was comparable to maximal stimulation of these cells with forskolin (2 x 10(-5) M). On the molecular level, there was a 10-fold increase in the expression of steroid acute regulatory peptide mRNA. This effect was independent of adipose angiotensin II as revealed by the stimulatory effect of fat cell-conditioned medium even in the presence of the angiotensin type 1 receptor antagonist, valsartan. None of the recently defined adipocytokines accounted for the effect. Mineralocorticoid-stimulating activity was heat sensitive and could be blunted by heating fat cell-conditioned medium to 99 degrees C. Centrifugal filtration based on molecular mass revealed at least two releasing factors: a heat sensitive fraction (molecular mass >50 kDa) representing 60% of total activity, and an inactive fraction (molecular mass <50 kDa). However, the recovery rate increased to 92% when combining these two fractions, indicating the interaction of at least two factors. In conclusion, human adipocytes secrete potent mineralocorticoid-releasing factors, suggesting a direct link between obesity and hypertension.

Differential regulation of endogenous glucose-6-phosphatase and phosphoenolpyruvate carboxykinase gene expression by the forkhead transcription factor FKHR in H4IIE-hepatoma cells.

The insulin responsive H4IIEC3 rat hepatoma cell line (H4 cells) was used in order to determine the role of the transcription factor FKHR in the regulation of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Both PEPCK and G6Pase contain putative FKHR binding sites in their promoter sequence. Using a retroviral expression system, we stably overexpressed FKHR in H4-cells. FKHR was phosphorylated in a PI 3-kinase- and Akt-dependent manner, and was translocated from the nucleus to the cytoplasm in response to insulin. Furthermore, overexpression of FKHR markedly increased the expression of the catalytic subunit of G6Pase (basal about 2.5-fold, dexamethasone/cAMP stimulated about fivefold, respectively). In contrast, both basal and dexamethasone/cAMP-induced levels of PEPCK mRNA were unaffected by FKHR-overexpression. These data suggest a specific function for FKHR in the regulation of hepatic gluconeogenesis at the level of G6Pase, but not PEPCK gene expression.

Phorbol ester-induced activation of mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase and extracellular-signal-regulated protein kinase decreases glucose-6-phosphatase gene expression.

Glucose-6-phosphatase (G6Pase) plays a central role in blood glucose homoeostasis, and insulin suppresses G6Pase gene expression by the activation of phosphoinositide 3-kinase (PI 3-kinase). Here, we show that the phorbol ester PMA decreases both basal and dexamethasone/cAMP-induced expression of a luciferase gene under the control of the G6Pase promoter in transiently transfected H4IIE hepatoma cells. This regulation was suppressed by the inhibitors of the mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK), PD98059 and U0126, but not by the inhibitor of PI 3-kinase, LY294002. The co-expression of a constitutively active mutant of MEK mimicked the regulation of G6Pase promoter activity by PMA. The effect of PMA on both basal and induced G6Pase gene transcription was impaired by the overexpression of a dominant negative MEK construct, as well as by the expression of mitogen-activated protein kinase phosphatase-1. The mutation of the forkhead-binding sites within the insulin-response unit of the G6Pase promoter, which decreases the effect of insulin on G6Pase gene expression, did not alter the regulation of gene expression by PMA. The data show that PMA decreases G6Pase gene expression by the activation of MEK and extracellular-signal regulated protein kinase. With that, PMA mimics the effect of insulin on G6Pase gene expression by a different signalling pathway.

The kinase DYRK1A phosphorylates the transcription factor FKHR at Ser329 in vitro, a novel in vivo phosphorylation site.

Forkhead in rhabdomyosarcoma (FKHR) is a transcription factor that has been implicated in the control of gene expression by insulin, as well as the regulation of apoptosis by survival factors. These signals trigger the protein kinase B (PKB)-catalysed phosphorylation of FKHR at three residues (Thr(24), Ser(256) and Ser(319)) by a phosphoinositide 3-kinase-dependent pathway that results in the nuclear exit and inactivation of this transcription factor. Here, we have identified a conserved residue (Ser(329)) as a novel in vivo phosphorylation site on FKHR. Ser(329) phosphorylation also decreases the ability of FKHR to stimulate gene transactivation and reduces the proportion of FKHR present in the nucleus. However, unlike the residues targetted by PKB, Ser(329) is phosphorylated in unstimulated HEK-293 cells, and phosphorylation is not increased by stimulation with insulin-like growth factor-1 or by transfection with 3-phosphoinositide-dependent protein kinase-1. We have also purified a protein kinase to near homogeneity from rabbit skeletal muscle that phosphorylates FKHR at Ser(329) specifically and identified it as DYRK1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A). We find that FKHR and DYRK1A co-localize in discrete regions of the nucleus and can be co-immunoprecipitated from cell extracts. These experiments suggest that DYRK1A may phosphorylate FKHR at Ser(329) in vivo.

Glucose deprivation induces Akt-dependent synthesis and incorporation of GLUT1, but not of GLUT4, into the plasma membrane of 3T3-L1 adipocytes.

Reduction of the glucose concentration in the culture medium of 3T3-L1 adipose cells below 1.25 mM produces a 4-8-fold stimulation of 2-deoxyglucose uptake which starts after a lag phase of 2 h and is maximal after 10-16 h. In the present study, we employed the 'membrane sheet assay' in order to re-assess the contribution of the transporter isoforms GLUT1 and GLUT4 to this effect. Immunochemical assay of glucose transporters in membranes prepared with the 'sheet assay' revealed that the effect reflected a marked increase of GLUT1 in the plasma membrane with no effect on GLUT4. Glucose deprivation increased the total cellular GLUT1 protein in parallel with the transport activity, whereas GLUT4 was unaltered. The specific PI 3-kinase inhibitor wortmannin inhibited the effect of glucose deprivation on transport activity and also on GLUT1 synthesis. Glucose deprivation produced a moderate, biphasic increase in the activity of the protein kinase Akt/PKB that was inhibitable by wortmannin. When wortmannin was added after stimulation of cells in order to assess the internalization rate of transporters, the effect of insulin was reversed considerably faster (T1/2 = 18 min) than that of glucose deprivation (T1/2 > 60 min). These data are consistent with the conclusion that the effect of glucose deprivation reflects a specific, Akt-dependent de-novo synthesis of GLUT1, and not of GLUT4, and its insertion into a plasma membrane compartment which is distinct from that of the insulin-sensitive GLUT1.

Regulation of GLUT1 gene transcription by the serine/threonine kinase Akt1.

We used mouse hepatoma (Hepa1c1c7) cells to study the role of the serine/threonine kinase Akt in the induction of GLUT1 gene expression. In order to selectively turn on the Akt kinase cascade, we expressed a hydroxytamoxifen-regulatable form of Akt (myristoylated Akt1 estrogen receptor chimera (MER-Akt1)) in the Hepa1c1c7 cells; we verified that hydroxytamoxifen stimulates MER-Akt1 activity to a similar extent as the activation of endogenous Akt by insulin. Our studies reveal that stimulation of MER-Akt1 by hydroxytamoxifen induces GLUT1 mRNA and protein accumulation to levels comparable to that induced by insulin; therefore, activation of the Akt cascade suffices to induce GLUT1 gene expression in this cell system. Furthermore, expression of a kinase-inactive Akt mutant partially inhibits the response of the GLUT1 gene to insulin. Additional studies reveal that the induction of GLUT1 mRNA by Akt and by insulin reflects increased mRNA synthesis and not decreased mRNA degradation. Our findings imply that the GLUT1 gene responds to insulin at the transcriptional level and that Akt mediates a step in the activation of GLUT1 gene expression in this system.

Up-regulation of Akt3 in estrogen receptor-deficient breast cancers and androgen-independent prostate cancer lines.

We measured the insulin-stimulated amount of Akt1, Akt2, and Akt3 enzymatic activities in four breast cancer cell lines and three prostate cancer cell lines. In the estrogen receptor-deficient breast cancer cells and the androgen-insensitive prostate cells, the amount of Akt3 enzymatic activity was approximately 20-60-fold higher than in the cells that were estrogen- or androgen-responsive. In contrast, the levels of Akt1 and -2 were not increased in these cells. The increase in Akt3 enzyme activity correlated with an increase in both Akt3 mRNA and protein. In a prostate cancer cell line lacking the tumor suppressor PTEN (a lipid and protein phosphatase), the basal enzymatic activity of Akt3 was constitutively elevated and represented the major active Akt in these cells. Finally, reverse transcription-PCR was used to examine the Akt3 expression in 27 primary breast carcinomas. The expression levels of Akt3 were significantly higher in the estrogen receptor-negative tumors in comparison to the estrogen receptor-positive tumors. To see if the increase in Akt3 could be due to chromosomal abnormalities, the Akt3 gene was assigned to human chromosome 1q44 by fluorescence in situ hybridization and radiation hybrid cell panel analyses. These results indicate that Akt3 may contribute to the more aggressive clinical phenotype of the estrogen receptor-negative breast cancers and androgen-insensitive prostate carcinomas.

Activation of protein kinase B/Akt is sufficient to repress the glucocorticoid and cAMP induction of phosphoenolpyruvate carboxykinase gene.

A rat hepatoma cell line, H4IIE, was stably transfected with a tamoxifen regulatable Akt-1 construct. Treatment of these cells with tamoxifen caused a rapid stimulation of Akt enzymatic activity that was comparable with the activity observed with the endogenous Akt after insulin stimulation. Prior studies have extensively documented that insulin can repress the glucocorticoid and cAMP-stimulated increase in phosphoenolpyruvate carboxykinase (PEPCK) gene transcription. Activation of this regulatable Akt with tamoxifen was found to mimic the dominant inhibitory effect of insulin on PEPCK gene transcription. Dose response curves to insulin and tamoxifen demonstrated that this response was very sensitive to Akt activation although the maximal response observed with tamoxifen activation was slightly less than that observed with insulin, indicating that the response to insulin may also involve other signaling cascades. The regulation of PEPCK transcription via Akt was, like that previously described for insulin, not dependent upon 70 kDa S6 kinase activity in that it was not inhibited by rapamycin. Finally, the expression of a kinase dead Akt was able to partially inhibit the ability of insulin to stimulate this response. In summary, the present results indicate that activation of Akt alone is sufficient to repress the glucocorticoid and cAMP-stimulated increase in PEPCK gene transcription.