Prodrug delivery of novel PTP1B inhibitors to enhance insulin signalling.

A growing percentage of the population is resistant to two key hormones - insulin and leptin - as a result of increased obesity, often leading to significant health consequences such as type 2 diabetes. Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of signalling by both of these hormones, so that inhibitors of this enzyme may provide promise for correcting endocrine abnormalities in both diabetes and obesity. As with other tyrosine phosphatases, identification of viable drug candidates targeting PTP1B has been elusive because of the nature of its active site. Beginning with novel phosphotyrosine mimetics, we have designed some of the most potent PTP1B inhibitors. However, their highly acidic structures limit intrinsic permeability and pharmacokinetics. Ester prodrugs of these inhibitors improve their drug-like properties with the goal of delivering these nanomolar inhibitors to the cytoplasm of cells within target tissues. In addition to identifying prodrugs that is able to deliver active drugs into cells to inhibit PTP1B and increase insulin signalling, these compounds were further modified to gain a variety of cleavage properties for targeting activity in vivo. One such prodrug candidate improved insulin sensitivity in ob/ob mice, with lowered fasting blood glucose levels seen in the context of lowered fasting insulin levels following 4 days of intraperitoneal dosing. The results presented in this study highlight the potential for design of orally active drug candidates targeting PTP1B, while also delineating the considerable challenges remaining.

Administration of myostatin does not alter fat mass in adult mice.

AIM: Myostatin, a member of the TGF-beta superfamily, is produced by skeletal muscle and acts as a negative regulator of muscle mass. It has also been suggested that low-dose administration of myostatin (2 mug/day) in rodents can reduce fat mass without altering muscle mass. In the current study, we attempted to further explore the effects of myostatin on adipocytes and its potential to reduce fat mass, since myostatin administration could potentially be a useful strategy to treat obesity and its complications in humans. METHODS: Purified myostatin protein was examined for its effects on adipogenesis and lipolysis in differentiated 3T3-L1 adipocytes as well as for effects on fat mass in wild-type, myostatin null and obese mice. RESULTS: While myostatin was capable of inhibiting adipogenesis in 3T3-L1 cells, it did not alter lipolysis in fully differentiated adipocytes. Importantly, pharmacological administration of myostatin over a range of doses (2-120 mug/day) did not affect fat mass in wild-type or genetically obese (ob/ob, db/db) mice, although muscle mass was significantly reduced at the highest myostatin dose. CONCLUSIONS: Our results suggest that myostatin does not reduce adipose stores in adult animals. Contrary to prior indications, pharmacological administration of myostatin does not appear to be an effective strategy to treat obesity in vivo.

Essential role for Gab2 in the allergic response.

Dos/Gab family scaffolding adapters (Dos, Gab1, Gab2) bind several signal relay molecules, including the protein-tyrosine phosphatase Shp-2 and phosphatidylinositol-3-OH kinase (PI(3)K); they are also implicated in growth factor, cytokine and antigen receptor signal transduction. Mice lacking Gab1 die during embryogenesis and show defective responses to several stimuli. Here we report that Gab2-/- mice are viable and generally healthy; however, the response (for example, degranulation and cytokine gene expression) of Gab2-/- mast cells to stimulation of the high affinity immunoglobulin-epsilon (IgE) receptor Fc(epsilon)RI is defective. Accordingly, allergic reactions such as passive cutaneous and systemic anaphylaxis are markedly impaired in Gab2-/- mice. Biochemical analyses reveal that signalling pathways dependent on PI(3)K, a critical component of Fc(epsilon)RI signalling, are defective in Gab2-/- mast cells. Our data identify Gab2 as the principal activator of PI(3)K in response to Fc(epsilon)RI activation, thereby providing genetic evidence that Dos/Gab family scaffolds regulate the PI(3)K pathway in vivo. Gab2 and/or its associated signalling molecules may be new targets for developing drugs to treat allergy.

Overexpression of the LAR (leukocyte antigen-related) protein-tyrosine phosphatase in muscle causes insulin resistance.

Previous reports indicate that the expression and/or activity of the protein-tyrosine phosphatase (PTP) LAR are increased in insulin-responsive tissues of obese, insulin-resistant humans and rodents, but it is not known whether these alterations contribute to the pathogenesis of insulin resistance. To address this question, we generated transgenic mice that overexpress human LAR, specifically in muscle, to levels comparable to those reported in insulin-resistant humans. In LAR-transgenic mice, fasting plasma insulin was increased 2.5-fold compared with wild-type controls, whereas fasting glucose was normal. Whole-body glucose disposal and glucose uptake into muscle in vivo were reduced by 39-50%. Insulin injection resulted in normal tyrosyl phosphorylation of the insulin receptor and insulin receptor substrate 1 (IRS-1) in muscle of transgenic mice. However, phosphorylation of IRS-2 was reduced by 62%, PI3' kinase activity associated with phosphotyrosine, IRS-1, or IRS-2 was reduced by 34-57%, and association of p85alpha with both IRS proteins was reduced by 39-52%. Thus, overexpression of LAR in muscle causes whole-body insulin resistance, most likely due to dephosphorylation of specific regulatory phosphotyrosines on IRS proteins. Our data suggest that increased expression and/or activity of LAR or related PTPs in insulin target tissues of obese humans may contribute to the pathogenesis of insulin resistance.

Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice.

Protein-tyrosine phosphatase 1B (PTP-1B) is a major protein-tyrosine phosphatase that has been implicated in the regulation of insulin action, as well as in other signal transduction pathways. To investigate the role of PTP-1B in vivo, we generated homozygotic PTP-1B-null mice by targeted gene disruption. PTP-1B-deficient mice have remarkably low adiposity and are protected from diet-induced obesity. Decreased adiposity is due to a marked reduction in fat cell mass without a decrease in adipocyte number. Leanness in PTP-1B-deficient mice is accompanied by increased basal metabolic rate and total energy expenditure, without marked alteration of uncoupling protein mRNA expression. In addition, insulin-stimulated whole-body glucose disposal is enhanced significantly in PTP-1B-deficient animals, as shown by hyperinsulinemic-euglycemic clamp studies. Remarkably, increased insulin sensitivity in PTP-1B-deficient mice is tissue specific, as insulin-stimulated glucose uptake is elevated in skeletal muscle, whereas adipose tissue is unaffected. Our results identify PTP-1B as a major regulator of energy balance, insulin sensitivity, and body fat stores in vivo.

Mice mutant for Egfr and Shp2 have defective cardiac semilunar valvulogenesis.

Atrioventricular and semilunar valve abnormalities are common birth defects, but how cardiac valvulogenesis is directed remains largely unknown. During studies of genetic interaction between Egfr, encoding the epidermal growth factor receptor, and Ptpn11, encoding the protein-tyrosine-phosphatase Shp2, we discovered that Egfr is required for semilunar, but not atrioventricular, valve development. Although unnoticed in earlier studies, mice homozygous for the hypomorphic Egfr allele waved-2 (Egfrwa2/wa2) exhibit semilunar valve enlargement resulting from over-abundant mesenchymal cells. Egfr-/- mice (CD1 background) have similar defects. The penetrance and severity of the defects in Egfrwa2/wa2 mice are enhanced by heterozygosity for a targeted mutation of exon 2 of Ptpn11 (ref. 3). Compound (Egfrwa2/wa2:Ptpn11+/-) mutant mice also show premature lethality. Electrocardiography, echocardiography and haemodynamic analyses showed that affected mice develop aortic stenosis and regurgitation. Our results identify the Egfr and Shp2 as components of a growth-factor signalling pathway required specifically for semilunar valvulogenesis, support the hypothesis that Shp2 is required for Egfr signalling in vivo, and provide an animal model for aortic valve disease.

Detection of the latent form of Epstein-Barr virus DNA in the peripheral blood of healthy individuals.

Epstein-Barr virus infects resting B cells in vitro and activates them to continuously proliferating lymphoblasts. Activation is essential for the virus to convert its linear genome to the covalently closed circular episomal form in which it persists in proliferating cells. However, in vivo, Epstein-Barr virus persists in resting B cells. We found that in these cells also the virus is present as an episome, suggesting that the cells must, at some time, have been activated and then returned to a resting state. This is the first direct demonstration, for any herpesvirus, of this form of the viral genome in normal persistently infected tissue. Since no linear viral DNA was detected, we estimate that fewer than 1 in 40 cells replicates the virus in the peripheral blood of healthy donors.

Deletional mapping of fifteen mouse VH gene families reveals a common organization for three Igh haplotypes.

In addition to the content of germ-line variable gene segments, the organization of V genes has been implicated in the development of the Ab repertoire. We have searched the expressed VH genes of BALB/c mice for additional VH gene families and utilized deletion mapping to explore the extent of VH gene family interspersion. We have identified and characterized one new VH gene family (VH15) and extended our previous studies of the Igha and Ighb haplotypes to include a third haplotype (Ighj) using a newly developed panel of pre-B cell lines (CXCB cell lines). We conclude that the Igha, Ighb, and Ighj haplotypes have a similar Igh-V locus structure. A refined deletional map for 15 VH gene families and an individual member of the VHSM7 family (H10) has been constructed based on the deletion profiles of 72 rearranged heavy chain loci. These results demonstrate previously unrecognized examples of interspersion among members of the VHS107, VH10, and VHSM7 families.

Characterization of the CD48 gene demonstrates a positive element that is specific to Epstein-Barr virus-immortalized B-cell lines and contains an essential NF-kappa B site.

Epstein-Barr virus (EBV) infection of mature, resting B cells drives them to become lymphoblasts expressing high levels of cell surface molecules, such as CD48, characteristically expressed on normal activated B cells. Here, we report on the identification of an enhancer element in the CD48 gene which reproducibly confers strong transcriptional activity only in EBV-positive B-lymphoblastoid cell lines. The element is not activated upon infection of established EBV-negative B-cell lines, indicating that EBV fails to drive these cells to a fully lymphoblastoid phenotype. An NF-kappa B binding site is an essential component of the element but alone is not sufficient to account for the activity or the specificity of the element. We have detected a specific nuclear protein complex that binds to the element and show that NF-kappa B1 (p50) is a part of this complex. The EBV-encoded latent membrane protein 1 is capable of transactivating the isolated CD48 NF-kappa B site but not the intact element, suggesting that the latent membrane protein 1-driven activation of NF-kappa B/Rel must interact with other regulatory pathways to control expression of cellular genes as EBV drives resting B cells into the cell cycle.

Is there a unique episome in EBV transformed B cells?

We have examined the frequency of episome formation in resting B cells 36 hr after infection with Epstein-Barr virus. We have detected an average of 0.65 episomal genomes per cell (SD 0.17). We have used this information to predict the distribution of circularization events in B cell clones derived from such infections. We have observed that in 32 out of 33 cases (with one unresolved) the B cell clone has arisen as a consequence of a single circularization event. This result is highly statistically different from the predicted distribution. We suggest that there is an exclusion or selection mechanism operating that favors either the formation or retention of a single circular genome per cell early in infection.

The prototypical Epstein-Barr virus-transformed lymphoblastoid cell line IB4 is an unusual variant containing integrated but no episomal viral DNA.

IB4 is a prototype, latently Epstein-Barr virus (EBV)-infected, lymphoblastoid cell line. We show here that IB4 contains only integrated EBV genomes. Episomal EBV DNA is not detected by Gardella gel analysis or in situ hybridization. Restriction enzyme mapping indicates that the EBV genomes first circularized and then integrated into and deleted part of the BamHI C fragment. IB4 is therefore the only lymphoblastoid cell line described to date that lacks episomal EBV and has integrated EBV genomes with joined ends. Thus, the detection of joined EBV termini on Southern blots is not as reliable as the Gardella gel system for detecting episomal EBV DNA, and IB4 is not an ideal prototype cell line for the study of latent infection by EBV.