Overexpression of protein-tyrosine phosphatase-1B in adipocytes inhibits insulin-stimulated phosphoinositide 3-kinase activity without altering glucose transport or Akt/Protein kinase B activation.

Previous studies suggested that protein-tyrosine phosphatase 1B (PTP1B) antagonizes insulin action by catalyzing dephosphorylation of the insulin receptor (IR) and/or other key proteins in the insulin signaling pathway. In adipose tissue and muscle of obese humans and rodents, PTP1B expression is increased, which led to the hypothesis that PTP1B plays a role in the pathogenesis of insulin resistance. Consistent with this, mice in which the PTP1B gene was disrupted exhibit increased insulin sensitivity. To test whether increased expression of PTP1B in an insulin-sensitive cell type could contribute to insulin resistance, we overexpressed wild-type PTP1B in 3T3L1 adipocytes using adenovirus-mediated gene delivery. PTP1B expression was increased approximately 3-5-fold above endogenous levels at 16 h, approximately 14-fold at 40 h, and approximately 20-fold at 72 h post-transduction. Total protein-tyrosine phosphatase activity was increased by 50% at 16 h, 3-4-fold at 40 h, and 5-6-fold at 72 h post-transduction. Compared with control cells, cells expressing high levels of PTP1B showed a 50-60% decrease in maximally insulin-stimulated tyrosyl phosphorylation of IR and insulin receptor substrate-1 (IRS-1) and phosphoinositide 3-kinase (PI3K) activity associated with IRS-1 or with phosphotyrosine. Akt phosphorylation and activity were unchanged. Phosphorylation of p42 and p44 MAP kinase (MAPK) was reduced approximately 32%. Overexpression of PTP1B had no effect on basal, submaximally or maximally (100 nm) insulin-stimulated glucose transport or on the EC(50) for transport. Our results suggest that: 1) insulin stimulation of glucose transport in adipocytes requires

Targeting of constitutively active phosphoinositide 3-kinase to GLUT4-containing vesicles in 3T3-L1 adipocytes.

Constitutive activation of phosphoinositide 3-kinase (PI3K) stimulates glucose transport and GLUT4 glucose transporter translocation to the plasma membrane in adipocytes. To determine whether a direct interaction of PI3K with GLUT4-containing vesicles (hereafter called GLUT4 vesicles) is important for the effect of insulin on GLUT4 translocation, we targeted constitutively active PI3K to GLUT4 vesicles. We fused the inter-Src homology region 2 of the regulatory p85alpha subunit of PI3K (iSH2) either to a C-terminal sequence of GLUT4 (G4c, amino acids 406-509) or to this region and the N-terminal tail of GLUT4 (G4n, amino acids 1-19), resulting in the fusion proteins iSH2-G4c and G4n-iSH2-G4c, respectively. Coexpression of the fusion proteins or untargeted iSH2 with the catalytic p110alpha subunit of PI3K (p110) in 3T3-L1 adipocytes by adenovirus-mediated gene transfer increased total PI3K activity in homogenates 5.0-6.7-fold over nontransduced cells or cells transduced with adenovirus encoding beta-galactosidase. In contrast, PI3K activity in GLUT4 vesicles increased 11-13-fold with expression of either targeted construct and p110 but only 2-fold with the untargeted iSH2 and p110, indicating successful targeting of PI3K to GLUT4 vesicles. Both targeted and nontargeted constructs stimulated DNA synthesis to levels greater than insulin, demonstrating that both types of constructs had biologic activity in intact cells. Despite this, untargeted iSH2/p110 coexpression was more effective in stimulating 2-deoxyglucose uptake (6-fold) than either iSH2-G4c/p110 or G4n-iSH2-G4c/p110 coexpression (both 2-fold). Only iSH2/p110 coexpression led to a significant GLUT4 translocation to the plasma membrane. Insulin-stimulated glucose transport was unaffected by any construct. Thus, a direct interaction between PI3K and GLUT4 vesicles is either not required or not sufficient for GLUT4 translocation and stimulation of glucose transport.

Hepatic glucose disposition during concomitant portal glucose and amino acid infusions in the dog.

The effect of concomitant intraportal infusion of glucose and gluconeogenic amino acids (AA) on net hepatic glucose uptake (NHGU) and glycogen synthesis was examined in 42-h-fasted dogs. After a basal period, there was a 240-min experimental period during which somatostatin was infused continuously into a peripheral vein and insulin and glucagon (at 3-fold basal and basal rates, respectively) and glucose (18.3 mumol.kg-1.min-1) were infused intraportally. One group (PoAA, n = 7) received an AA mixture intraportally at 7.6 mumol.kg-1.min-1, whereas the other group (NoAA, n = 6) did not receive AA. Arterial blood glucose concentrations and hepatic glucose loads were the same in the two groups. NHGU averaged 4.8 +/- 2.0 (PoAA) and 9.4 +/- 2.0 (NoAA) mumol.kg-1.min-1 (P < 0.05), and tracer-determined hepatic glucose uptake was 4.6 +/- 1.6 (PoAA) and 10.0 +/- 1.7 (NoAA) mumol.kg-1.min-1 (P < 0.05). AA data for PoAA and NoAA, respectively, were as follows: arterial blood concentrations, 1,578 +/- 133 vs. 1,147 +/- 86 microM (P < 0.01); hepatic loads, 56 +/- 3 vs. 32 +/- 4 mumol.kg-1.min-1 (P < 0.01); and net hepatic uptakes, 14.1 +/- 1.4 vs. 5.6 +/- 0.4 mumol.kg-1.min-1 (P < 0.01). The rate of net hepatic glycogen synthesis was 7.5 +/- 1.9 (PoAA) vs. 10.7 +/- 2.3 (NoAA) mumol.kg-1.min-1 (P = 0.1). In a net sense, intraportal gluconeogenic amino acid delivery directed glucose carbon away from the liver. Despite this, net hepatic carbon uptake was equivalent in the presence and absence of amino acid infusion.

CLS advanced degrees and career enhancement. Part 1--Comparison of career data.

OBJECTIVE: Determine whether recipients of clinical laboratory science (CLS) advanced degrees (MS) experience greater career achievements than their baccalaureate level (BS) colleagues. DESIGN: Two similar questionnaires were used-one for certified or licensed CLS professionals who had earned advanced CLS degrees (MS); the other for matched BS CLS colleagues. SETTING: Five academic programs that conduct both National Accrediting Agency for Clinical Laboratory Sciences accredited CLS education and CLS MS degree programs participated. PARTICIPANTS: The number of survey respondents was 220, 117 with advanced CLS degrees and 103 BS level controls. There were 99 matched pairs, i.e., 198 individuals were matched for gender, residence region, and years of experience. MAIN OUTCOME MEASURES: Careers of BS vs. MS respondents were statistically compared, e.g., fractions with managerial level jobs, relative earnings increases per year, numbers of publications and reports, and other professional contributions. RESULTS: Compared to their BS degree controls, MS degree respondents had more managerial level jobs (62% MS; 36% BS), a higher frequency of job change (once per 4.3 years MS; once per 5.9 years BS), and a higher increase per year of earnings (9.1% MS; 8.1% BS). A greater percentage of the MS degree graduates (77%) than the BS level controls (33%) had authored external publications; the responses related to authorship of institutional reports and procedures were less different-84% MS and 64% BS. Professional contributions to their institutions or profession were cited slightly more frequently by the MS graduates (65%) than by the BS level controls (55%). CONCLUSION: Compared to their matched BS level CLS colleagues, CLS MS degree recipients had greater job mobility, greater management authority, higher salary, and more numerous professional contributions.

CLS advanced degrees and career enhancement. Part 2--A comparison of perceptions.

OBJECTIVE: To determine whether recipients of clinical laboratory science (CLS) advanced degrees (MS) perceive greater career enhancement value related to earning an advanced degree than is perceived by their baccalaureate level (BS) colleagues. DESIGN: Two questionnaires were used-one for certified or licensed CLS professionals who had earned MS CLS degrees; the other for matched BS CLS colleagues. SETTING: Five academic programs that conduct both National Accrediting Agency for Clinical Laboratory Sciences accredited CLS education and CLS MS degree programs participated. PARTICIPANTS: The number of survey respondents was 220 (117-MS; 103-BS level controls). The groups were matched for gender, residence region, and years of experience. MAIN OUTCOME MEASURES: The primary outcome measurements were the perceived benefits of having a CLS MS degree, the reasons for and against obtaining a CLS MS degree, and the overall evaluation of CLS degree programs at both levels. RESULTS: The highest perceived benefit of having a CLS MS degree was the same in both groups, "enhanced self esteem and confidence". The highest priority motivation of MS degree recipients for obtaining a CLS advanced degree was "personal satisfaction". The highest priority reason of the BS group for not obtaining a CLS advanced degree was "family obligation". In both levels of degree programs the subject most commonly cited as needing modification was laboratory management. CONCLUSION: The results indicate that CLS professionals who have CLS MS degrees perceive a greater career enhancement value of advanced CLS degrees than their BS level colleagues.

Retroviral transfer of a bacterial alkyltransferase gene (ada) into human bone marrow cells protects against O6-benzylguanine plus 1, 3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity.

The antitumor activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is limited by the O6-alkylguanine-DNA alkyltransferase (ATase) in tumor cells and by delayed myelosuppression. Inactivation of neoplastic ATase by O6-benzylguanine (BG) improves the therapeutic index for BCNU. We have demonstrated previously that BG + BCNU-induced myelosuppression in mice is reduced by expression of the BG-resistant ATase ada in murine bone marrow. We have now generated an amphotropic retrovirus containing the ada gene and tested the effectiveness of ada expression in preventing BG + BCNU cytotoxicity in human hematopoietic progenitor cells. A retroviral producer clone with a biological titer of 6.5 x 10(4) colony-forming units/ml and 4.4 pmol ATase/mg protein was used for transduction of bone marrow. Cocultivation of these ada producer cells with progenitor cells from six normal individuals resulted in 1.9-3. 9-fold protection against BG + BCNU-induced cytotoxicity in committed progenitor cell assays. Furthermore, this cytoprotective effect was associated with a high transduction efficiency (40%) and a 2-fold increase of ATase activity in the surviving committed progenitor cell colonies. These data provide a basis for testing the clinical effectiveness of retroviral ada gene transfer into hematopoietic cells to increase the therapeutic index of BG + BCNU.

Neural and pancreatic influences on net hepatic glucose uptake and glycogen synthesis.

The role of the liver nerves in the disposition of peripherally administered glucose was examined in seven hepatic innervated (HI) and nine hepatic denervated (HD) 42-h-fasted conscious dogs. After a 40-min basal period, there was a 4-h experimental period during which the hepatic glucose load was increased twofold via peripheral glucose infusion. Somatostatin was infused to suppress pancreatic endocrine secretion, and insulin and glucagon were infused intraportally to produce a fourfold increase in insulin and a gradual decrease (approximately 25%) in glucagon. The area under the curve of net hepatic glucose uptake (NHGU) during the glucose infusion period totaled 483 +/- 82 and 335 +/- 32 mg/kg in HD and HI, respectively (P < 0.05). The area under the curve of the hepatic fractional extraction of glucose was 27% greater in HD (P < 0.05). Net hepatic lactate output was similar in the two groups, and net hepatic glycogen synthesis was 3.8 +/- 0.8 vs. 2.7 +/- 0.5 mg.kg dog wt-1.min-1 in HD and HI, respectively (P = 0.13). The direct pathway of glycogen synthesis was responsible for 54-58% of net hepatic glycogen synthesis in both HI and HD (n = 6 for both). In summary 1) NHGU in response to peripheral glucose infusion was approximately 44% greater in HD than in HI, 2) net hepatic glycogen synthesis was enhanced by 41% in HD although the probability of this change was 0.13, and 3) the contribution of the direct pathway to glycogen synthesis was the same in HD and HI. These data are consistent with a role for the liver nerves in regulating the magnitude of NHGU in response to glucose administration. They also indicate that the absence of liver nerves may reduce glycogen turnover during glucose infusion.

Changes in O6-methylguanine-DNA methyltransferase expression during immortalization of cloned human fibroblasts.

Suppressed expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), characterized as the Mer- phenotype, occurs only in malignant or transformed cell lines. To investigate the relationship between the transformation process and loss of MGMT expression, we derived 20 cloned lines of IMR90 normal fibroblasts transfected with the plasmid pSV3neo expressing the SV40 large-T antigen. Of the five lines that were grown until crisis phase, four emerged as continuously proliferating immortal lines. Of these, only one retained MGMT, the other three having become Mer-. In every case the loss of MGMT coincided with the final phase of immortalization following crisis. Because these were cloned cell lines it is clear that the phenotypic change to Mer- is not merely due to selection of a Mer- cell from the initial population, but must involve a cellular change in MGMT regulation. It is not clear if increased mutation rate associated with loss of MGMT results in increased frequency of an immortalization event or if an immortalization event, such as telomere disruption, results in MGMT suppression. In addition, we have shown that, consistent with previous observations, both hypermethylation in promoter sequences and hypomethylation of downstream sequences in the body of the gene were closely associated with loss of MGMT expression. These studies also illustrate the utility of these new cloned cell lines for characterizing molecular events associated with transformation and immortalization.

From chart tracking to workflow management.

The current interest in system-wide integration appears to be based on the assumption that an organization, by digitizing information and accepting a common standard for the exchange of such information, will improve the accessibility of this information and automatically experience benefits resulting from its more productive use. We do not dispute this reasoning, but assert that an organization's capacity for effective change is proportional to the understanding of the current structure among its personnel. Our workflow manager is based on the use of a Parameterized Petri Net (PPN) model which can be configured to represent an arbitrarily detailed picture of an organization. The PPN model can be animated to observe the model organization in action, and the results of the animation analyzed. This simulation is a dynamic ongoing process which changes with the system and allows members of the organization to pose "what if" questions as a means of exploring opportunities for change. We present, the "workflow management system" as the natural successor to the tracking program, incorporating modeling, scheduling, reactive planning, performance evaluation, and simulation. This workflow management system is more than adequate for meeting the needs of a paper chart tracking system, and, as the patient record is computerized, will serve as a planning and evaluation tool in converting the paper-based health information system into a computer-based system.

Effects of information feedback and pulse oximetry on the incidence of anesthesia complications.

No standard outcome measures exist to evaluate the effect of interventions intended to improve the quality of anesthesia care. The authors established a clinically practical definition of outcome, and used it to assess the effect of feedback of information about complications and the effect of pulse oximetry on the rate and severity of important anesthesia-related problems encountered in the operating room (OR) and recovery room (RR). On admission to the RR, the patient's anesthetist documented Recovery-Room-Impact Events (RRIE), defined as an "unanticipated, undesirable, possibly anesthesia-related effect that required intervention, was pertinent to recovery-room care, and did or could cause at least moderate morbidity." Following a control period with no feedback of data, intense feedback of grouped (anonymous) RRIE rates was provided. Later, pulse oximeters were introduced to all anesthetizing locations. Among 12,088 patients (71% of all RR admissions), 18% had at least one RRIE in the OR or RR. The most common RRIEs were hypotension (4.4%), arrhythmia (3.9%), hypertension (1.5%), intubation difficulties (0.8%), hypoventilation (0.8%), and hypovolemia (0.6%). Feedback of information produced no demonstrable change in the rate of RRIEs. Although significantly fewer patients experienced RRIEs (15.6% vs. 12.4%, P less than 0.0001), hypotensive RRIEs (5.2% vs. 3.8%, P = 0.0003), and hypovolemic RRIEs (0.88% vs. 0.42%, P = 0.0017) following the introduction of pulse oximetry in the OR, confounding factors prevent establishment of a cause-and-effect relationship. Quality assurance may require more direct intervention and individual feedback to be effective. Still, the RRIE measure requires minimal effort at low cost and encourages improved transmission of information at the time of admission to recovery-room care.