Preclinical proof of concept for VivoVec, a lentiviral-based platform for in vivo CAR T-cell engineering.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation. METHODS: UB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice. RESULTS: In vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy. CONCLUSIONS: These findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing.

Stochastic fate decisions of HSCs after transplantation: early contribution, symmetric expansion, and pool formation.

Hematopoietic stem cells (HSCs) are assumed to be rare, infrequently dividing, long-lived cells not involved in immediate recovery after transplantation. Here, we performed unprecedented high-density clonal tracking in nonhuman primates and found long-term persisting HSC clones to actively contribute during early neutrophil recovery, and to be the main source of blood production as early as 50 days after transplantation. Most surprisingly, we observed a rapid decline in the number of unique HSC clones, while persisting HSCs expanded, undergoing symmetric divisions to create identical siblings and formed clonal pools ex vivo as well as in vivo. In contrast to the currently assumed model of hematopoietic reconstitution, we provide evidence for contribution of HSCs in short-term recovery as well as symmetric expansion of individual clones into pools. These findings provide novel insights into HSC biology, informing the design of HSC transplantation and gene therapy studies.

Efficient polymer nanoparticle-mediated delivery of gene editing reagents into human hematopoietic stem and progenitor cells.

Clinical applications of hematopoietic stem cell (HSC) gene editing are limited due to their complex and expensive logistics. HSC editing is commonly performed ex vivo using electroporation and requires good manufacturing practice (GMP) facilities, similar to bone marrow transplant centers. In vivo gene editing could overcome this limitation; however, electroporation is unsuitable for systemic in vivo applications to HSCs. Here we evaluated polymer-based nanoparticles (NPs), which could also be used for in vivo administration, for the delivery of mRNA and nucleases to human granulocyte colony-stimulating factor (GCSF)-mobilized CD34+ cells. NP-mediated ex vivo delivery showed no toxicity, and the efficiency was directly correlated with the charge of the NPs. In a side-by-side comparison with electroporation, NP-mediated gene editing allowed for a 3-fold reduction in the amount of reagents, with similar efficiency. Furthermore, we observed enhanced engraftment potential of human HSCs in the NSG mouse xenograft model using NPs. Finally, mRNA- and nuclease-loaded NPs were successfully lyophilized for storage, maintaining their transfection potential after rehydration. In conclusion, we show that polymer-based NP delivery of mRNA and nucleases has the potential to overcome current limitations of HSC gene editing. The predictable transfection efficiency, low toxicity, and ability to lyophilize NPs will greatly enhance the portability and provide a highly promising platform for HSC gene therapy.

The Role of Behavioral and Neurocognitive Functioning in Substance Use Among Youth with Perinatally Acquired HIV Infection and Perinatal HIV Exposure Without Infection.

This study examined associations of self-regulatory behavior and cognitive functioning with substance use (SU) to inform interventions for youth with perinatal HIV infection (YPHIV) or exposure but uninfected (YPHEU). Youth aged 7-15 years (YPHIV, n = 390; YPHEU, n = 211) were followed longitudinally with cognitive testing and behavioral questionnaires including self-report of alcohol, marijuana, tobacco, and other SU. Cox proportional hazards analyses were used to examine correlates of initiating each substance for those without prior use at baseline and generalized estimating equation analyses were used to address associations of cognitive/behavioral measurements with SU prevalence for the entire sample. Lower self-reported self-regulation skills, but higher cognitive functioning abilities, were associated with initiation and prevalent use of alcohol and marijuana regardless of HIV status. Our findings suggest SU screening tools and self-regulation interventions developed for general adolescent populations should be implemented for those with PHIV, who may be at heightened risk for SU-related health consequences.

RESUMEN: En este estudio se examina el vínculo del comportamiento autorregulado y la función cognoscitiva con el consumo de sustancias para argumentar intervenciones para los jóvenes con infección perinatal por el VIH (JIPVIH) y los jóvenes con exposición perinatal sin infección por el VIH (JEPSIVIH). Se hizo un seguimiento longitudinal de jóvenes de 7 a 15 años de edad (JIPVIH, n = 390; JEPSIVIH, n = 211) por medio de pruebas cognoscitivas y cuestionarios sobre el comportamiento, incluyendo el autoinforme de consumo de alcohol, marihuana, tabaco y otras sustancias. Se usaron los análisis Cox de riesgos proporcionales para examinar factores correlacionados con el inicio del consumo de cada sustancia por personas no consumidoras en el punto de referencia inicial. Asimismo, se usaron análisis de ecuaciones de estimación generalizadas para examinar la asociación entre la prevalencia del consumo de sustancias y las medidas cognoscitivas y las medidas conductuales para toda la muestra. Habilidades de autorregulación disminuidas, según autoinforme, pero capacidades superiores de función cognoscitiva, fueron vinculadas con el inicio y consumo frecuente de alcohol y marihuana, independientemente de la condición de VIH. Nuestros hallazgos sugieren que herramientas para detectar el consumo de sustancias e intervenciones de autorregulación creadas para la población general de adolescentes se deberían implementar para los JIPVIH que podrían correr mayores riesgos de sufrir consecuencias en la salud relacionadas con el consumo de sustancias.

Isolation of a Highly Purified HSC-enriched CD34+CD90+CD45RA- Cell Subset for Allogeneic Transplantation in the Nonhuman Primate Large-animal Model.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a common treatment for patients suffering from different hematological disorders. Allo-HCT in combination with hematopoietic stem cell (HSC) gene therapy is considered a promising treatment option for millions of patients with HIV+ and acute myeloid leukemia. Most currently available HSC gene therapy approaches target CD34-enriched cell fractions, a heterogeneous mix of mostly progenitor cells and only very few HSCs with long-term multilineage engraftment potential. As a consequence, gene therapy approaches are currently limited in their HSC targeting efficiency, very expensive consuming huge quantities of modifying reagents, and can lead to unwanted side effects in nontarget cells. We have previously shown that purified CD34+CD90+CD45RA- cells are enriched for multipotent HSCs with long-term multilineage engraftment potential, which can reconstitute the entire hematopoietic system in an autologous nonhuman primate transplant model. Here, we tested the feasibility of transplantation with purified CD34+CD90+CD45RA- cells in the allogeneic setting in a nonhuman primate model. METHODS: To evaluate the feasibility of this approach, CD34+CD90+CD45RA- cells from 2 fully major histocompatibility complex-matched, full sibling rhesus macaques were sort-purified, quality controlled, and transplanted. Engraftment and donor chimerism were evaluated in the peripheral blood and bone marrow of both animals. RESULTS: Despite limited survival due to infectious complications, we show that the large-scale sort-purification and transplantation of CD34+CD90+CD45RA- cells is technically feasible and leads to rapid engraftment of cells in bone marrow in the allogeneic setting and absence of cotransferred T cells. CONCLUSIONS: We show that purification of an HSC-enriched CD34+ subset can serve as a potential stem cell source for allo-HCTs. Most importantly, the combination of allo-HCT and HSC gene therapy has the potential to treat a wide array of hematologic and nonhematologic disorders.

Purification of Human CD34+CD90+ HSCs Reduces Target Cell Population and Improves Lentiviral Transduction for Gene Therapy.

Hematopoietic stem cell (HSC) gene therapy has the potential to cure many genetic, malignant, and infectious diseases. We have shown in a nonhuman primate gene therapy and transplantation model that the CD34+CD90+ cell fraction was exclusively responsible for multilineage engraftment and hematopoietic reconstitution. In this study, we show the translational potential of this HSC-enriched CD34 subset for lentivirus-mediated gene therapy. Alternative HSC enrichment strategies include the purification of CD133+ cells or CD38low/- subsets of CD34+ cells from human blood products. We directly compared these strategies to the isolation of CD90+ cells using a good manufacturing practice (GMP) grade flow-sorting protocol with clinical applicability. We show that CD90+ cell selection results in about 30-fold fewer target cells in comparison to CD133+ or CD38low/- CD34+ hematopoietic stem and progenitor cell (HSPC) subsets without compromising the engraftment potential in vivo. Single-cell RNA sequencing confirmed nearly complete depletion of lineage-committed progenitor cells in CD90+ fractions compared to alternative selections. Importantly, lentiviral transduction efficiency in purified CD90+ cells resulted in up to 3-fold higher levels of engrafted gene-modified blood cells. These studies should have important implications for the manufacturing of patient-specific HSC gene therapy and gene-engineered cell products.

Therapeutically relevant engraftment of a CRISPR-Cas9-edited HSC-enriched population with HbF reactivation in nonhuman primates.

Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34+CD90+CD45RA-, allowing for a 10-fold reduction in the number of transplanted target cells, thus considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases.

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells.

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

Contact guidance diversity in rotationally aligned collagen matrices.

Cancer cell metastasis is responsible for approximately 90% of deaths related to cancer. The migration of cancer cells away from the primary tumor and into healthy tissue is driven in part by contact guidance, or directed migration in response to aligned extracellular matrix. While contact guidance has been a focus of many studies, much of this research has explored environments that present 2D contact guidance structures. Contact guidance environments in 3D more closely resemble in vivo conditions and model cell-ECM interactions better than 2D environments. While most cells engage in directed migration on potent 2D contact guidance cues, there is diversity in response to contact guidance cues based on whether the cell migrates with a mesenchymal or amoeboid migration mode. In this paper, rotational alignment of collagen gels was used to study the differences in contact guidance between MDA-MB-231 (mesenchymal) and MTLn3 (amoeboid) cells. MDA-MB-231 cells migrate with high directional fidelity in aligned collagen gels, while MTLn3 cells show no directional migration. The collagen stiffness was increased through glycation, resulting in decreased MDA-MB-231 directionality in aligned collagen gels. Interestingly, partial inhibition of cell contractility dramatically decreased directionality in MDA-MB-231 cells. The directionality of MDA-MB-231 cells was most sensitive to ROCK inhibition, but unlike in 2D contact guidance environments, cell directionality and speed are more tightly coupled. Modulation of the contractile apparatus appears to more potently affect contact guidance than modulation of extracellular mechanical properties of the contact guidance cue. STATEMENT OF SIGNIFICANCE: Collagen fiber alignment in the tumor microenvironment directs migration, a process called contact guidance, enhancing the efficiency of cancer invasion and metastasis. 3D systems that assess contact guidance by locally orienting collagen fiber alignment are lacking. Furthermore, cell type differences and the role of extracellular matrix stiffness in tuning contact guidance fidelity are not well characterized. In this paper rotational alignment of collagen fibers is used as a 3D contact guidance cue to illuminate cell type differences and the role of extracellular matrix stiffness in guiding cell migration along aligned fibers of collagen. This local alignment offers a simple approach by which to couple collagen alignment with gradients in other directional cues in devices such as microfluidic chambers.

Differences in predictors of cervical and breast cancer screening by screening need in uninsured Latino women.

BACKGROUND: Latino women experience higher mortality for cervical cancer and lower 5-year survival for breast cancer than non-Latino White women. Adherence with screening recommendations can increase chances of survival, yet the factors that influence screening behaviors in uninsured women are not well documented. METHODS: Uninsured Latino women (N = 467) recruited in four US cities participated in the study. Logistic regression was used to model adherence to recommendations by screening type (cervical or breast cancer) and screening need (needs to obtain initial screening, overdue for rescreening, up-to-date with rescreening). RESULTS: Predictors differed by type of screening and screening need. Women who reported exposure to cancer education were more likely to have had a mammogram and to be up-to-date with Pap smear screening than women without such exposure. Women who were younger, had more than a sixth grade education, and/or had children were more likely to have had a Pap smear. Older women who had been in the US the longest were more likely to be overdue for a Pap smear. Women with incomes 5000 to 7000 were more likely to have obtained a mammogram. Regional differences were found with respect to mammography screening and maintenance behaviors. CONCLUSIONS: Exposure to cancer education is an important predictor of screenings among uninsured urban Latino women. The potential of creating educational interventions that can increase screening rates among women who evidence health disparities is encouraging. Recruitment strategies to reach women in need of screenings are provided.

Serum concentrations of resistin-like molecules beta and gamma are elevated in high-fat-fed and obese db/db mice, with increased production in the intestinal tract and bone marrow.

AIMS/HYPOTHESIS: Resistin and the resistin-like molecules (RELMs) comprise a novel class of cysteine-rich proteins. Among the RELMs, RELMbeta and RELMgamma are produced in non-adipocyte tissues, but the regulation of their expression and their physiological roles are largely unknown. We investigated in mice the tissue distribution and dimer formation of RELMbeta and RELMgamma and then examined whether their serum concentrations and tissue expression levels are related to insulin resistance. METHODS: Specific antibodies against RELMbeta and RELMgamma were generated. Dimer formation was examined using COS cells and the colon. RELMbeta and RELMgamma tissue localisation and expression levels were analysed by an RNase protection assay, immunoblotting and immunohistochemical study. Serum concentrations in high-fat-fed and db/db mice were also measured using the specific antibodies. RESULTS: The intestinal tract produces RELMbeta and RELMgamma, and colonic epithelial cells in particular express both RELMbeta and RELMgamma. In addition, RELMbeta and RELMgamma were shown to form a homodimer and a heterodimer with each other, in an overexpression system using cultured cells, and in mouse colon and serum. Serum RELMbeta and RELMgamma levels in high-fat-fed mice were markedly higher than those in mice fed normal chow. Serum RELMbeta and RELMgamma concentrations were also clearly higher in db/db mice than in lean littermates. Tissue expression levels revealed that elevated serum concentrations of RELMbeta and RELMgamma are attributable to increased production in the colon and bone marrow. CONCLUSIONS/INTERPRETATION: RELMbeta and RELMgamma form homo/heterodimers, which are secreted into the circulation. Serum concentrations of RELMbeta and RELMgamma may be a novel intestinal-tract-mediating regulator of insulin sensitivity, possibly involved in insulin resistance induced by obesity and a high-fat diet.

Oxidative stress induces insulin resistance by activating the nuclear factor-kappa B pathway and disrupting normal subcellular distribution of phosphatidylinositol 3-kinase.

AIMS/HYPOTHESIS: Oxidative stress is associated with diabetes, hypertension and atherosclerosis. Insulin resistance is implicated in the development of these disorders. We tested the hypothesis that oxidative stress induces insulin resistance in rats, and endeavoured to identify mechanisms linking the two. METHODS: Buthionine sulfoximine (BSO), an inhibitor of glutathione synthase, was administered to Sprague-Dawley rats and 3T3-L1 adipocytes. Glucose metabolism and insulin signalling both in vivo and in 3T3-L1 adipocytes were examined. In 3T3-L1 adipocytes, the effects of overexpression of a dominant negative mutant of inhibitory kappa B (I kappa B), one role of which is to block oxidative-stress-induced nuclear factor (NF)-kappa B activation, were investigated. RESULTS: In rats given BSO for 2 weeks, the plasma lipid hydroperoxide level doubled, indicating increased oxidative stress. A hyperinsulinaemic-euglycaemic clamp study and a glucose transport assay using isolated muscle and adipocytes revealed insulin resistance in BSO-treated rats. BSO treatment also impaired insulin-induced glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes. In BSO-treated rat muscle, adipose tissue and 3T3-L1 adipocytes, insulin-induced IRS-1 phosphorylation in the low-density microsome (LDM) fraction was specifically decreased, while that in whole cell lysates was not altered, and subsequent translocation of phosphatidylinositol (PI) 3-kinase from the cytosol and the LDM fraction was disrupted. BSO-induced impairments of insulin action and insulin signalling were reversed by overexpressing the dominant negative mutant of I kappa B, thereby suppressing NF-kappa B activation. CONCLUSIONS/INTERPRETATION: Oxidative stress induces insulin resistance by impairing IRS-1 phosphorylation and PI 3-kinase activation in the LDM fraction, and NF-kappa B activation is likely to be involved in this process.

Interindividual variation of serum haloperidol concentrations in Japanese patients--clinical considerations on steady-state serum level-dose ratios.

OBJECTIVE: Marked interpatient variability in haloperidol (HAL) level-dose (L/D) ratios makes it difficult to use the administered dose for predicting serum concentrations. OBJECTIVE: To investigate the effect of dose, age, total body weight and co-medication on steady-state HAL L/D ratios. METHOD: Retrospective analysis of dose and HAL blood level data from 168 patients. RESULTS: The HAL L/D ratio decreased curvilinearly with increasing daily dose of HAL. The patients treated with concomitant antiparkinsonian drugs showed a mean HAL L/D ratio that was 24.9% higher than those without antiparkinsonian drugs. The patients treated with concomitant antiepileptic drugs showed a mean HAL L/D ratio that was 27.2% lower than those without antiepileptic drugs. The mean HAL L/D ratio of patients treated with concomitant CYP2D6 substrates was not significantly different from those without CYP2D6 substrates. CONCLUSION: There is a wide interindividual variability in blood levels of HAL in patients given the same dose. Routine monitoring of HAL serum level is useful, especially in patients who require associated antiepileptic and/or antiparkinsonian medication.

Effects of N-alpha-methyl-histamine on human H(2) receptors expressed in CHO cells.

BACKGROUND: Production of N-alpha-methyl-histamine (NAMH), a histamine H(3) receptor (H3R) agonist, is reportedly promoted in Helicobacter pylori infected human gastric mucosa. NAMH was suggested to act directly on histamine H(2) receptors (H2Rs) in animals to stimulate acid secretion and to be a H2R agonist. As H2Rs and H3Rs play different roles in gastric acid secretion, it is very important to verify that NAMH is a H2R agonist. AIMS: To determine whether NAMH is a H2R agonist, as well as a H3R agonist. METHODS: We used a Chinese hamster ovary (CHO) cell line expressing human H2Rs (CHO-H2R) and control CHO cells. Expression of human H2Rs was confirmed by tiotidine binding. cAMP production in CHO-H2R and control cells in response to histamine or NAMH was measured. cAMP production in response to 10(-7) M NAMH was also measured in the presence or absence of the H2R antagonist famotidine and the H3R antagonist thioperamide. RESULTS: NAMH dose dependently stimulated cAMP productions in CHO-H2R cells. This production was inhibited by famotidine but not by thioperamide. Control CHO cells were unresponsive to either histamine or NAMH. In addition, the effect of NAMH, in terms of cAMP production in CHO-H2R cells, was more potent than that of histamine-that is, with a lower EC(50) concentration and higher maximal cAMP production. Both NAMH and histamine, but not R-alpha-methyl-histamine, effectively inhibited [(3)H] tiotidine binding to CHO-H2R cells. CONCLUSIONS: NAMH, which is produced in the gastric mucosa by H pylori, is a potent H2R agonist as well as a H3R agonist.

Unique phosphorylation mechanism of Gab1 using PI 3-kinase as an adaptor protein.

Grb2-associated binder-1 (Gab1) undergoes tyrosine phosphorylation in response to stimulation by growth factors and hormones including insulin, epidermal growth factor (EGF), nerve growth factor (NGF), and hepatocyte growth factor (HGF). However, the HGF receptor is the only one known to associate directly with Gab1. Herein, we explore the mechanism of Gab1 phosphorylation by other receptor protein-tyrosine kinases unable to bind to Gab1 directly. The Src homology 2 (SH2) domain of the phosphatidylinositol 3-kinase (PI3K) regulatory subunit binds Gab1 in a phosphorylation-independent manner. Moreover, the regulatory subunit of PI3K can mediate the association of Gab1 and receptor protein-tyrosine kinases including the insulin, EGF, and NGF receptors, all of which phosphorylate Gab1. Thus, it appears that the PI3K regulatory subunit acts as an adaptor protein via a phosphotyrosyl-independent SH2 interaction, allowing Gab1 to serve as a substrate for several tyrosine kinases. This is a new role for the PI3K regulatory subunit.

Regulation of phosphoinositide metabolism, Akt phosphorylation, and glucose transport by PTEN (phosphatase and tensin homolog deleted on chromosome 10) in 3T3-L1 adipocytes.

To investigate the roles of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in the regulation of 3-position phosphorylated phosphoinositide metabolism as well as insulin-induced Akt phosphorylation and glucose metabolism, wild-type PTEN and its phosphatase-dead mutant (C124S) with or without an N-terminal myristoylation tag were overexpressed in Sf-9 cells and 3T3-L1 adipocytes using baculovirus and adenovirus systems, respectively. When expressed in Sf-9 cells together with the p110alpha catalytic subunit of phosphoinositide 3-kinase, myristoylated PTEN markedly reduced the accumulations of both phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate induced by p110alpha. In contrast, overexpression of the C124S mutants apparently increased these accumulations. In 3T3-L1 adipocytes, insulin-induced accumulations of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate were markedly suppressed by overexpression of wild-type PTEN with the N-terminal myristoylation tag, but not by that without the tag. On the contrary, the C124S mutants of PTEN enhanced insulin-induced accumulations of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. Interestingly, the phosphorylation level of Akt at Thr308 (Akt2 at Thr309), but not at Ser473 (Akt2 at Ser474), was revealed to correlate well with the accumulation of phosphatidylinositol 3,4,5-trisphosphate modified by overexpression of these PTEN proteins. Finally, insulin-induced increases in glucose transport activity were significantly inhibited by the overexpression of myristoylated wild-type PTEN, but were not enhanced by expression of the C124S mutant of PTEN. Therefore, in conclusion, 1) PTEN dephosphorylates both phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in vivo, and the C124S mutants interrupt endogenous PTEN activity in a dominant-negative manner. 2) The membrane targeting process of PTEN may be important for exerting its function. 3) Phosphorylations of Thr309 and Ser474 of Akt2 are regulated differently, and the former is regulated very sensitively by the function of PTEN. 4) The phosphorylation level of Ser474, but not that of Thr309, in Akt2 correlates well with insulin-stimulated glucose transport activity in 3T3-L1 adipocytes. 5) The activity of endogenous PTEN may not play a major role in the regulation of glucose transport activity in 3T3-L1 adipocytes.

Palmitoylation of the canine histamine H2 receptor occurs at Cys(305) and is important for cell surface targeting.

To determine the presence and functional role of the histamine H2 receptor (H2R) palmitoylation, a receptor with a Cys(305) to Ala (A(305) receptor) mutation was generated. Wild-type (WT) and A(305) receptors were tagged at their N-termini with a hemagglutinin (HA) epitope. WT, but not A(305), receptors incorporated [3H]palmitate by metabolic labeling, indicating that the H2R is palmitoylated at Cys(305). Immunocytochemistry of WT and A(305) receptors expressed in COS7 cells revealed WT receptors to be distributed at the plasma membrane, while the majority of A(305) receptors were localized intracellularly with only a small portion being at the plasma membrane. However, the affinity of the A(305) receptor for tiotidine was comparable to that of the WT receptor. In addition, when the amounts of cell surface receptors as determined by anti-HA antibody binding were equivalent, A(305) receptors mediated production of more cAMP than WT receptors. Preincubation of COS7 cells expressing each receptor with 10(-5) M histamine for 30 min reduced subsequent cAMP production in response to histamine via the receptors to similar extents, indicating that palmitoylation is not necessary for desensitization. In addition, cell surface A(305) receptors were capable of being internalized from the cell surface at a rate and extent similar to those of WT receptors. Finally, CHO cell lines stably expressing either WT or A(305) receptors were incubated with 10(-5) M histamine for 1, 6, 12 and 24 h. Total amounts of WT and A(305) receptors, as determined by tiotidine binding, were reduced by incubation, indicating downregulation. Downregulation of the A(305) receptor was more extensive than that of the WT receptor. Thus, palmitoylation of the H2R might be important for targeting to the cell surface and stability.

Inhibitory effect of hyperglycemia on insulin-induced Akt/protein kinase B activation in skeletal muscle.

To determine the molecular mechanism underlying hyperglycemia-induced insulin resistance in skeletal muscles, postreceptor insulin-signaling events were assessed in skeletal muscles of neonatally streptozotocin-treated diabetic rats. In isolated soleus muscle of the diabetic rats, insulin-stimulated 2-deoxyglucose uptake, glucose oxidation, and lactate release were all significantly decreased compared with normal rats. Similarly, insulin-induced phosphorylation and activation of Akt/protein kinase B (PKB) and GLUT-4 translocation were severely impaired. However, the upstream signal, including phosphorylation of the insulin receptor (IR) and insulin receptor substrate (IRS)-1 and -2 and activity of phosphatidylinositol (PI) 3-kinase associated with IRS-1/2, was enhanced. The amelioration of hyperglycemia by T-1095, a Na(+)-glucose transporter inhibitor, normalized the reduced insulin sensitivity in the soleus muscle and the impaired insulin-stimulated Akt/PKB phosphorylation and activity. In addition, the enhanced PI 3-kinase activation and phosphorylation of IR and IRS-1 and -2 were reduced to normal levels. These results suggest that sustained hyperglycemia impairs the insulin-signaling steps between PI 3-kinase and Akt/PKB, and that impaired Akt/PKB activity underlies hyperglycemia-induced insulin resistance in skeletal muscle.

Insulin resistance with enhanced insulin signaling in high-salt diet-fed rats.

Previous clinical studies showed an apparent correlation between hypertension and insulin resistance, and patients with diabetes are known to have increased blood pressure responsiveness to salt loading. To investigate the effect of high salt intake on insulin sensitivity and the insulin signaling pathway, a high-salt diet (8% NaCl) or a normal diet was given to 7-week-old SD rats for 2 weeks. High salt-fed rats developed slightly but significantly higher systolic blood pressure than controls (133 +/- 2 vs. 117 +/- 2 mmHg, P < 0.001), with no change in food intake or body weight. High salt-fed rats were slightly hyperglycemic (108.5 +/- 2.8 vs. 97.8 +/- 2.5 mg/dl, P = 0.01) and slightly hyperinsulinemic (0.86 +/- 0.07 vs. 0.61 +/- 0.06 ng/ml, P = 0.026) in the fasting condition, as compared with controls. Hyperinsulinemic-euglycemic clamp study revealed a 52.7% decrease in the glucose infusion rate and a 196% increase in hepatic glucose production in high salt-fed rats, which also showed a 66.4% decrease in 2-deoxyglucose uptake into isolated skeletal muscle and a 44.5% decrease in insulin-induced glycogen synthase activation in liver, as compared with controls. Interestingly, despite the presence of insulin resistance, high salt-fed rats showed enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1, IRS-2 (liver and muscle), and IRS-3 (liver only). Phosphatidylinositol (PI) 3-kinase activities associated with IRS and phosphotyrosine in the insulin-stimulated condition increased 2.1- to 4.1-fold, as compared with controls. Insulin-induced phosphorylation of Ser-473 of Akt and Ser-21 of glycogen synthase kinase-3 also increased 2.9- and 2-fold, respectively, in the liver of the high salt-fed rats. Therefore, in both the liver and muscle of high salt-fed rats, intracellular insulin signaling leading to PI 3-kinase activation is enhanced and insulin action is attenuated. The hyperinsulinemic-euglycemic clamp study showed that decreased insulin sensitivity induced with a high-salt diet was not reversed by administration of pioglitazone. The following can be concluded: 1) a high-salt diet may be a factor promoting insulin resistance, 2) the insulin-signaling step impaired by high salt intake is likely to be downstream from PI 3-kinase or Akt activation, and 3) this unique insulin resistance mechanism may contribute to the development of diabetes in patients with hypertension.

MKK6/3 and p38 MAPK pathway activation is not necessary for insulin-induced glucose uptake but regulates glucose transporter expression.

p38 mitogen-activated protein kinase (MAPK), which is situated downstream of MAPK kinase (MKK) 6 and MKK3, is activated by mitogenic or stress-inducing stimuli, as well as by insulin. To clarify the role of the MKK6/3-p38 MAPK pathway in the regulation of glucose transport, dominant negative p38 MAPK and MKK6 mutants and constitutively active MKK6 and MKK3 mutants were overexpressed in 3T3-L1 adipocytes and L6 myotubes using an adenovirus-mediated transfection procedure. Constitutively active MKK6/3 mutants up-regulated GLUT1 expression and down-regulated GLUT4 expression, thereby significantly increasing basal glucose transport but diminishing transport induced by insulin. Similar effects were elicited by chronic (24 h) exposure to tumor necrosis factor alpha, interleukin-1beta, or 200 mm sorbitol, all activate the MKK6/3-p38 MAPK pathway. SB203580, a specific p38 MAPK inhibitor, attenuated these effects, further confirming that both MMK6 and MMK3 act via p38 MAPK, whereas they had no effect on the increase in glucose transport induced by a constitutively active MAPK kinase 1 (MEK1) mutant or by myristoylated Akt. In addition, suppression of p38 MAPK activation by overexpression of a dominant negative p38 MAPK or MKK6 mutant did not diminish insulin-induced glucose uptake by 3T3-L1 adipocytes. It is thus apparent that activation of p38 MAPK is not essential for insulin-induced increases in glucose uptake. Rather, p38 MAPK activation leads to a marked down-regulation of insulin-induced glucose uptake via GLUT4, which may underlie cellular stress-induced insulin resistance caused by tumor necrosis factor alpha and other factors.