The lasting effects of neuronal activity on brain development involve calcium-dependent gene expression. Using a strategy called transactivator trap, we cloned a calcium-responsive transactivator called CREST (for calcium-responsive transactivator). CREST is a SYT-related nuclear protein that interacts with adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB)-binding protein (CBP) and is expressed in the developing brain. Mice that have a targeted disruption of the crest gene are viable but display defects in cortical and hippocampal dendrite development. Cortical neurons from crest mutant mice are compromised in calcium-dependent dendritic growth. Thus, calcium activation of CREST-mediated transcription helps regulate neuronal morphogenesis.

Insulin maintains vascular smooth muscle cell (VSMC) quiescence yet can also promote VSMC migration. The mechanisms by which insulin exerts these contrasting effects were examined using ␣-smooth muscle actin (␣-SMA) as a marker of VSMC phenotype because ␣-SMA is highly expressed in quiescent but not migratory VSMC. Insulin alone maintained VSMC quiescence and modestly stimulated VSMC migration. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, decreased insulin-stimulated expression of ␣-SMA mRNA by 26% and protein by 48% but had no effect on VSMC migration. PD98059, a mitogen-activated protein kinase (MAPK) kinase inhibitor, decreased insulin-induced VSMC migration by 52% but did not affect ␣-SMA levels. Platelet-derived growth factor (PDGF) promoted dedifferentiation of VSMC, and insulin counteracted this effect. Furthermore, insulin increased ␣-SMA mRNA and protein levels to 111 and 118%, respectively, after PDGF-induced dedifferentiation, an effect inhibited by wortmannin. In conclusion, insulin's ability to maintain VSMC quiescence and reverse the dedifferentiating influence of PDGF is mediated via the PI3K pathway, whereas insulin promotes VSMC migration via the MAPK pathway. Thus, with impaired PI 3-kinase signaling and intact MAPK signaling, as seen in insulin resistance, insulin may lose its ability to maintain VSMC quiescence and instead promote VSMC migration.

Insulin sensitivity determines the effectiveness of dietary macronutrient composition on weight loss in obese women. Obes Res. 2005;13:703-709. Objective: To determine whether macronutrient composition of a hypocaloric diet can enhance its effectiveness and whether insulin sensitivity (Si) affects the response to hypocaloric diets. Research Methods and Procedures: Obese nondiabetic insulin-sensitive (fasting insulin Ͻ 10 U/mL; n ϭ 12) and obese nondiabetic insulin-resistant (fasting insulin Ͼ 15 U/mL; n ϭ 9) women (23 to 53 years old) were randomized to either a high carbohydrate (CHO) (HC)/low fat (LF) (60% CHO, 20% fat) or low CHO (LC)/high fat (HF) (40% CHO, 40% fat) hypocaloric diet. Primary outcome measures after a 16-week dietary intervention were: changes in body weight (BW), Si, resting metabolic rate, and fasting lipids. Results: Insulin-sensitive women on the HC/LF diet lost 13.5 Ϯ 1.2% (p Ͻ 0.001) of their initial BW, whereas those on the LC/HF diet lost 6.8 Ϯ 1.2% (p Ͻ 0.001; p Ͻ 0.002 between the groups). In contrast, among the insulin-resistant women, those on the LC/HF diet lost 13.4 Ϯ 1.3% (p Ͻ 0.001) of their initial BW as compared with 8.5 Ϯ 1.4% (p Ͻ 0.001) lost by those on the HC/LF diet (p Ͻ 0.04 between two groups). These differences could not be explained by changes in resting metabolic rate, activity, or intake. Overall, changes in Si were associated with the degree of weight loss (r ϭ Ϫ0.57, p Ͻ 0.05). Discussion: The state of Si determines the effectiveness of macronutrient composition of hypocaloric diets in obese women. For maximal benefit, the macronutrient composition of a hypocaloric diet may need to be adjusted to correspond to the state of Si.

Insulin resistance is a cardinal feature of normal pregnancy and excess growth hormone (GH) states, but its underlying mechanism remains enigmatic. We previously found a significant increase in the p85 regulatory subunit of phosphatidylinositol kinase (PI 3-kinase) and striking decrease in IRS-1-associated PI 3-kinase activity in the skeletal muscle of transgenic animals overexpressing human placental growth hormone. Herein, using transgenic mice bearing deletions in p85␣, p85␤, or insulin-like growth factor-1, we provide novel evidence suggesting that overexpression of p85␣ is a primary mechanism for skeletal muscle insulin resistance in response to GH. We found that the excess in total p85 was entirely accounted for by an increase in the free p85␣-specific isoform. In mice with a liverspecific deletion in insulin-like growth factor-1, excess GH caused insulin resistance and an increase in skeletal muscle p85␣, which was completely reversible using a GH-releasing hormone antagonist. To understand the role of p85␣ in GH-induced insulin resistance, we used mice bearing deletions of the genes coding for p85␣ or p85␤, respectively (p85␣ ؉/؊ and p85␤ ؊/؊ ). Wild type and p85␤؊/؊ mice developed in vivo insulin resistance and demonstrated overexpression of p85␣ and reduced insulin-stimulated PI 3-kinase activity in skeletal muscle in response to GH. In contrast, p85␣ ؉/؊ mice retained global insulin sensitivity and PI 3-kinase activity associated with reduced p85␣ expression. These findings demonstrated the importance of increased p85␣ in mediating skeletal muscle insulin resistance in response to GH and suggested a potential role for reducing p85␣ as a therapeutic strategy for enhancing insulin sensitivity in skeletal muscle.Insulin resistance is a common feature associated with growth hormone excess; however, the cellular mechanism underlying insulin resistance remains elusive. We previously demonstrated that transgenic mice overexpressing human placental growth hormone (TG-hPGH), 2 at levels comparable with the third trimester of pregnancy, were severely insulin-resistant and display increased amounts of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) in skeletal muscle (1).Several recent studies have suggested that a disrupted balance between the levels of the PI 3-kinase subunits may alter insulin-stimulated PI 3-kinase activity (2-6). This enzyme consists of a regulatory subunit, p85, and a catalytic subunit, p110 (7). Normally, the regulatory subunit exists in stoichiometric excess to the catalytic one, resulting in a pool of free p85 monomers not associated with the p110 catalytic subunit. The p85 monomers bind to phosphorylated IRS proteins, blocking access to p85-p110 heterodimers. Thus, there exists a balance between the free p85 monomer and the p85-p110 heterodimer with the latter being responsible for the PI 3-kinase activity. Increases or decreases in expression of p85 shift this balance in favor of either free p85 or p85-p110 complexes (3-6). Because the monomer and the heterod...

Aims/hypothesis: We sought to define early molecular alterations associated with nutritionally induced insulin resistance in humans. Methods: Insulin sensitivity was assessed using a hyperinsulinaemic-euglycaemic clamp in eight healthy women while on an isocaloric diet and after 3 days of overfeeding (50% above eucaloric diet). Expression of phosphatidylinositol (PI) 3-kinase subunits p85α and p110 was assessed and measurements were made of IRS-1-associated PI 3-kinase activity, tyrosine and serine phosphorylation of IRS-1, and serine and threonine phosphorylation of p70S6 kinase. Measurements were made in skeletal muscle biopsies obtained before and after overfeeding. Results: Three days of overfeeding resulted in a reduction of insulin sensitivity accompanied by: (1) increased expression of skeletal muscle p85α; (2) an alteration in the ratio of p85α to p110; (3) a decrease in the amount of IRS-1-associated p110; and (4) a decrease in PI 3-kinase activity. Increases in expression of p85α and in the p85α:p110 ratio demonstrated a highly significant inverse correlation with insulin sensitivity, and changes in PI 3-kinase activity correlated with changes in insulin sensitivity. Tyrosine and serine phosphorylation of IRS-1 and serine and threonine phosphorylation of p70S6 kinase were unaffected by 3 days of overfeeding. Conclusions/interpretation: We identified a novel mechanism of nutritionally induced insulin resistance in healthy women of normal weight. We conclude that increased expression of p85α may be one of the earliest molecular alterations in the mechanism of the insulin resistance associated with overfeeding.

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Refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T) is a provisional entity in the current World Health Organization classification and is thought to be a myelodysplastic/myeloproliferative neoplasm (MDS/MPN). We analyzed 18 cases of RARS-T. All patients had thrombocytosis (platelet count, 515-1,100 × 10(3)/μL [515-1,100 × 10(9)/L]) and anemia (hemoglobin level, 7.2-12.6 g/dL [72-126 g/L]). Three patients had mild leukocytosis (WBC count, 3,900-16,300/μL [3.9-16.3 × 10(9)/L]). Ring sideroblasts were 8% to 75% in the bone marrow. Megakaryocytes showed a spectrum of morphologic findings. JAK2(V617F) was identified in 9 of 15 cases, including 7 of 9 with thrombocytosis (platelet count, >600 × 10(3)/μL [600 × 10(9)/L]) and 1 with 8% ring sideroblasts. The MPL(W515L) mutation was not detected (n = 9). We conclude that RARS-T is a pathogenetically heterogeneous group of limited diagnostic usefulness. Approximately 60% of cases carry JAK2(V617F)and seem to be closer to an MPN in which ring sideroblasts may be a secondary phenomenon. The remaining cases usually lack the JAK2(V617F)mutation, have a platelet count less than 600 × 10(3)/μL (600 × 10(9)/L), and may represent an MDS or MPN with thrombocytosis of unknown mechanisms.

Background The value of assessing CD5 expression in the differential diagnosis of small B-cell neoplasms is well established. Usually CD5 is assessed qualitatively. Materials and Methods We assessed CD5 expression levels by quantitative flow cytometry immunophenotyping to determine possible differences among various small B-cell neoplasms. We performed 4-color flow cytometry analysis on peripheral blood (PB) and bone marrow (BM) aspirate specimens and quantified CD5 expression in various mature small B-cell lymphomas and leukemias. We also assessed CD5 levels in PB samples of normal donors (controls). Results Cases of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma had higher levels of CD5 compared with control (benign) B-cells (p < 0.001). Cases of marginal zone lymphoma (MZL) and hairy cell leukemia (HCL) had CD5 levels similar to control B-cells (p > 0.05), while cases of follicular lymphoma (FL) and lymphoplasmacytic lymphoma (LPL) had significantly lower CD5 levels than control B-cells (p <0.05). In B-cell neoplasms, a high level of CD5 expression correlated with a homogeneous pattern of positive events whereas lower CD5 levels correlated with a heterogeneous pattern of positive events. Conclusions Using flow cytometric immunophenotypic analysis to quantify CD5 levels can aid in diagnosis. CD5 expression levels are substantially higher in CLL and mantle cell lymphoma and expression is observed in a homogeneous pattern as compared with other B-cell neoplasms that are either negative for CD5 or express this antigen at lower levels with a heterogeneous pattern of expression. However, there is some overlap in CD5 expression levels between a subset of atypical CLL and MZL cases.