High growth hormone serum partially protects mice against Trypanosoma cruzi infection.

Chagas disease (CD) is one of the most devasting parasitic diseases in the Americas, affecting 7-8 million people worldwide. In vitro and in vivo experiments have demonstrated that growth hormone (GH) serum levels decrease as CD progresses. Interestingly, inactivating mutations in the GH receptor in humans result in Laron syndrome (LS), a clinical entity characterized by increased serum levels of GH and decreased insulin growth factor-1 (IGF-1). The largest cohort of LS subjects lives in the southern provinces of Ecuador. Remarkably, no clinical CD cases have been reported in these individuals despite living in highly endemic areas. In the current ex vivo study, we employed serum from GHR-/- mice, also known as LS mice (a model of GH resistance with high GH and low IGF-1 levels), and serum from bovine GH (bGH) transgenic mice (high GH and IGF-1), to test the effect on Trypanosoma cruzi infection. We infected mouse fibroblast L-cells with T. cruzi (etiological CD infectious agent) and treated them with serum from each mouse type. Treatment with GHR-/- serum (LS mice) significantly decreased L-cell infection by 28% compared with 48% from control wild-type mouse serum (WT). Treatment with bGH mouse serum significantly decreased infection of cells by 41% compared with 54% from WT controls. Our results suggest that high GH and low IGF-1 in blood circulation, as typically seen in LS individuals, confer partial protection against T. cruzi infection. This study is the first to report decreased T. cruzi infection using serum collected from two modified mouse lines with altered GH action (GHR-/- and bGH).

Growth Hormone Alters Circulating Levels of Glycine and Hydroxyproline in Mice.

Growth hormone (GH) has established effects on protein metabolism, such as increasing protein synthesis and decreasing amino acid degradation, but its effects on circulating amino acid levels are less studied. To investigate this relationship, metabolomic analyses were used to measure amino acid concentrations in plasma and feces of mice with alterations to the GH axis, namely bovine GH transgenic (bGH; increased GH action) and GH receptor knockout (GHRKO; GH resistant) mice. To determine the effects of acute GH treatment, GH-injected GH knockout (GHKO) mice were used to measure serum glycine. Furthermore, liver gene expression of glycine metabolism genes was assessed in bGH, GHRKO, and GH-injected GHKO mice. bGH mice had significantly decreased plasma glycine and increased hydroxyproline in both sexes, while GHRKO mice had increased plasma glycine in both sexes and decreased hydroxyproline in males. Glycine synthesis gene expression was decreased in bGH mice (Shmt1 in females and Shmt2 in males) and increased in GHRKO mice (Shmt2 in males). Acute GH treatment of GHKO mice caused decreased liver Shmt1 and Shmt2 expression and decreased serum glycine. In conclusion, GH alters circulating glycine and hydroxyproline levels in opposing directions, with the glycine changes at least partially driven by decreased glycine synthesis.

Disruption of Growth Hormone Receptor in Adipocytes Improves Insulin Sensitivity and Lifespan in Mice.

Growth hormone receptor knockout (GHRKO) mice have been used for 25 years to uncover some of the many actions of growth hormone (GH). Since they are extremely long-lived with enhanced insulin sensitivity and protected from multiple age-related diseases, they are often used to study healthy aging. To determine the effect that adipose tissue has on the GHRKO phenotype, our laboratory recently created and characterized adipocyte-specific GHRKO (AdGHRKO) mice, which have increased adiposity but appear healthy with enhanced insulin sensitivity. To test the hypothesis that removal of GH action in adipocytes might partially replicate the increased lifespan and healthspan observed in global GHRKO mice, we assessed adiposity, cytokines/adipokines, glucose homeostasis, frailty, and lifespan in aging AdGHRKO mice of both sexes. Our results show that disrupting the GH receptor gene in adipocytes improved insulin sensitivity at advanced age and increased lifespan in male AdGHRKO mice. AdGHRKO mice also exhibited increased fat mass, reduced circulating levels of insulin, c-peptide, adiponectin, resistin, and improved frailty scores with increased grip strength at advanced ages. Comparison of published mean lifespan data from GHRKO mice to that from AdGHRKO and muscle-specific GHRKO mice suggests that approximately 23% of lifespan extension in male GHRKO is due to GHR disruption in adipocytes vs approximately 19% in muscle. Females benefited less from GHR disruption in these 2 tissues with approximately 19% and approximately 0%, respectively. These data indicate that removal of GH's action, even in a single tissue, is sufficient for observable health benefits that promote long-term health, reduce frailty, and increase longevity.

Growth hormone receptor antagonism downregulates ATP-binding cassette transporters contributing to improved drug efficacy against melanoma and hepatocarcinoma in vivo.

Knockdown of GH receptor (GHR) in melanoma cells in vitro downregulates ATP-binding cassette-containing (ABC) transporters and sensitizes them to anti-cancer drug treatments. Here we aimed to determine whether a GHR antagonist (GHRA) could control cancer growth by sensitizing tumors to therapy through downregulation of ABC transporters in vivo. We intradermally inoculated Fluc-B16-F10 mouse melanoma cells into GHA mice, transgenic for a GHR antagonist (GHRA), and observed a marked reduction in tumor size, mass and tumoral GH signaling. Moreover, constitutive GHRA production in the transgenic mice significantly improved the response to cisplatin treatment by suppressing expression of multiple ABC transporters and sensitizing the tumors to the drug. We confirmed that presence of a GHRA and not a mere absence of GH is essential for this chemo-sensitizing effect using Fluc-B16-F10 allografts in GH knockout (GHKO) mice, where tumor growth was reduced relative to that in GH-sufficient controls but did not sensitize the tumor to cisplatin. We extended our investigation to hepatocellular carcinoma (HCC) using human HCC cells in vitro and a syngeneic mouse model of HCC with Hepa1-6 allografts in GHA mice. Gene expression analyses and drug-efflux assays confirm that blocking GH significantly suppresses the levels of ABC transporters and improves the efficacy of sorafenib towards almost complete tumor clearance. Human patient data for melanoma and HCC show that GHR RNA levels correlate with ABC transporter expression. Collectively, our results validate in vivo that combination of a GHRA with currently available anti-cancer therapies can be effective in attacking cancer drug resistance.

Mice with gene alterations in the GH and IGF family.

Much of our understanding of GH's action stems from animal models and the generation and characterization of genetically altered or modified mice. Manipulation of genes in the GH/IGF1 family in animals started in 1982 when the first GH transgenic mice were produced. Since then, multiple laboratories have altered mouse DNA to globally disrupt Gh, Ghr, and other genes upstream or downstream of GH or its receptor. The ability to stay current with the various genetically manipulated mouse lines within the realm of GH/IGF1 research has been daunting. As such, this review attempts to consolidate and summarize the literature related to the initial characterization of many of the known gene-manipulated mice relating to the actions of GH, PRL and IGF1. We have organized the mouse lines by modifications made to constituents of the GH/IGF1 family either upstream or downstream of GHR or to the GHR itself. Available data on the effect of altered gene expression on growth, GH/IGF1 levels, body composition, reproduction, diabetes, metabolism, cancer, and aging are summarized. For the ease of finding this information, key words are highlighted in bold throughout the main text for each mouse line and this information is summarized in Tables 1, 2, 3 and 4. Most importantly, the collective data derived from and reported for these mice have enhanced our understanding of GH action.

Growth hormone receptor gene disruption in mature-adult mice improves male insulin sensitivity and extends female lifespan.

Studies in multiple species indicate that reducing growth hormone (GH) action enhances healthy lifespan. In fact, GH receptor knockout (GHRKO) mice hold the Methuselah prize for the world's longest-lived laboratory mouse. We previously demonstrated that GHR ablation starting at puberty (1.5 months), improved insulin sensitivity and female lifespan but results in markedly reduced body size. In this study, we investigated the effects of GHR disruption in mature-adult mice at 6 months old (6mGHRKO). These mice exhibited GH resistance (reduced IGF-1 and elevated GH serum levels), increased body adiposity, reduced lean mass, and minimal effects on body length. Importantly, 6mGHRKO males have enhanced insulin sensitivity and reduced neoplasms while females exhibited increased median and maximal lifespan. Furthermore, fasting glucose and oxidative damage was reduced in females compared to males irrespective of Ghr deletion. Overall, disrupted GH action in adult mice resulted in sexual dimorphic effects suggesting that GH reduction at older ages may have gerotherapeutic effects.

Pharmacologic targeting of Cdc42 GTPase by a small molecule Cdc42 activity-specific inhibitor prevents platelet activation and thrombosis.

Gene targeting of Cdc42 GTPase has been shown to inhibit platelet activation. In this study, we investigated a hypothesis that inhibition of Cdc42 activity by CASIN, a small molecule Cdc42 Activity-Specific INhibitor, may down regulate platelet activation and thrombus formation. We investigated the effects of CASIN on platelet activation in vitro and thrombosis in vivo. In human platelets, CASIN, but not its inactive analog Pirl7, blocked collagen induced activation of Cdc42 and inhibited phosphorylation of its downstream effector, PAK1/2. Moreover, addition of CASIN to washed human platelets inhibited platelet spreading on immobilized fibrinogen. Treatment of human platelets with CASIN inhibited collagen or thrombin induced: (a) ATP secretion and platelet aggregation; and (b) phosphorylation of Akt, ERK and p38-MAPK. Pre-incubation of platelets with Pirl7, an inactive analog of CASIN, failed to inhibit collagen induced aggregation. Washing of human platelets after incubation with CASIN eliminated its inhibitory effect on collagen induced aggregation. Intraperitoneal administration of CASIN to wild type mice inhibited ex vivo aggregation induced by collagen but did not affect the murine tail bleeding times. CASIN administration, prior to laser-induced injury in murine cremaster muscle arterioles, resulted in formation of smaller and unstable thrombi compared to control mice without CASIN treatment. These data suggest that pharmacologic targeting of Cdc42 by specific and reversible inhibitors may lead to the discovery of novel antithrombotic agents.

Growth Hormone Upregulates Mediators of Melanoma Drug Efflux and Epithelial-to-Mesenchymal Transition In Vitro and In Vivo.

Growth hormone (GH) and the GH receptor (GHR) are expressed in a wide range of malignant tumors including melanoma. However, the effect of GH/insulin-like growth factor (IGF) on melanoma in vivo has not yet been elucidated. Here we assessed the physical and molecular effects of GH on mouse melanoma B16-F10 and human melanoma SK-MEL-30 cells in vitro. We then corroborated these observations with syngeneic B16-F10 tumors in two mouse lines with different levels of GH/IGF: bovine GH transgenic mice (bGH; high GH, high IGF-1) and GHR gene-disrupted or knockout mice (GHRKO; high GH, low IGF-1). In vitro, GH treatment enhanced mouse and human melanoma cell growth, drug retention and cell invasion. While the in vivo tumor size was unaffected in both bGH and GHRKO mouse lines, multiple drug-efflux pumps were up regulated. This intrinsic capacity of therapy resistance appears to be GH dependent. Additionally, epithelial-to-mesenchymal transition (EMT) gene transcription markers were significantly upregulated in vivo supporting our current and recent in vitro observations. These syngeneic mouse melanoma models of differential GH/IGF action can be valuable tools in screening for therapeutic options where lowering GH/IGF-1 action is important.

The Effects of 20-kDa Human Placental GH in Male and Female GH-deficient Mice: An Improved Human GH?

A rare 20K isoform of GH-V (here abbreviated as GHv) was discovered in 1998. To date, only 1 research article has characterized this isoform in vivo, observing that GHv treatment in male high-fat fed rats had several GH-like activities, but unlike GH lacked diabetogenic and lactogenic activities and failed to increase IGF-1 or body length. Therefore, the current study was conducted to further characterize the in vivo activities of GHv in a separate species and in a GH-deficient model (GH-/- mice) and with both sexes represented. GHv-treated GH-/- mice had significant increases to serum IGF-1, femur length, body length, body weight, and lean body mass and reduced body fat mass similar to mice receiving GH treatment. GH treatment increased circulating insulin levels and impaired insulin sensitivity; in contrast, both measures were unchanged in GHv-treated mice. Since GHv lacks prolactin receptor (PRLR) binding activity, we tested the ability of GH and GHv to stimulate the proliferation of human cancer cell lines and found that GHv has a decreased proliferative response in cancers with high PRLR. Our findings demonstrate that GHv can stimulate insulin-like growth factor-1 and subsequent longitudinal body growth in GH-deficient mice similar to GH, but unlike GH, GHv promoted growth without inhibiting insulin action and without promoting the growth of PRLR-positive cancers in vitro. Thus, GHv may represent improvements to current GH therapies especially for individuals at risk for metabolic syndrome or PRLR-positive cancers.

Growth Hormone Upregulates Melanocyte-Inducing Transcription Factor Expression and Activity via JAK2-STAT5 and SRC Signaling in GH Receptor-Positive Human Melanoma.

Growth hormone (GH) facilitates therapy resistance in the cancers of breast, colon, endometrium, and melanoma. The GH-stimulated pathways responsible for this resistance were identified as suppression of apoptosis, induction of epithelial-to-mesenchymal transition (EMT), and upregulated drug efflux by increased expression of ATP-binding cassette containing multidrug efflux pumps (ABC-transporters). In extremely drug-resistant melanoma, ABC-transporters have also been reported to mediate drug sequestration in intracellular melanosomes, thereby reducing drug efficacy. Melanocyte-inducing transcription factor (MITF) is the master regulator of melanocyte and melanoma cell fate as well as the melanosomal machinery. MITF targets such as the oncogene MET, as well as MITF-mediated processes such as resistance to radiation therapy, are both known to be upregulated by GH. Therefore, we chose to query the direct effects of GH on MITF expression and activity towards conferring chemoresistance in melanoma. Here, we demonstrate that GH significantly upregulates MITF as well as the MITF target genes following treatment with multiple anticancer drug treatments such as chemotherapy, BRAF-inhibitors, as well as tyrosine-kinase inhibitors. GH action also upregulated MITF-regulated processes such as melanogenesis and tyrosinase activity. Significant elevation in MITF and MITF target gene expression was also observed in mouse B16F10 melanoma cells and xenografts in bovine GH transgenic (bGH) mice compared to wild-type littermates. Through pathway inhibitor analysis we identified that both the JAK2-STAT5 and SRC activities were critical for the observed effects. Additionally, a retrospective analysis of gene expression data from GTEx, NCI60, CCLE, and TCGA databases corroborated our observed correlation of MITF function and GH action. Therefore, we present in vitro, in vivo, and in silico evidence which strongly implicates the GH-GHR axis in inducing chemoresistance in human melanoma by driving MITF-regulated and ABC-transporter-mediated drug clearance pathways.

GH Knockout Mice Have Increased Subcutaneous Adipose Tissue With Decreased Fibrosis and Enhanced Insulin Sensitivity.

In 1997, our laboratory used targeted gene disruption of the GH receptor (GHR) to generate GHR knockout (GHR-/-) mice, which have been used in >127 published studies to help elucidate GH's numerous activities. However, because GH replacement studies cannot be performed using this line, a GH knockout mouse line via targeted disruption of the GH gene is needed. Therefore, we created and characterized GH gene-disrupted (GH-/-) mice. GH-/- mice have severely decreased IGF-1 levels, small body size, and altered body composition with increased adiposity. GH-/- mice are extremely insulin sensitive but glucose intolerant, with a dramatic reduction in pancreatic islet size. Importantly, disruption of the GH gene had profound and depot-specific effects on white adipose tissue (WAT). Subcutaneous WAT from male and female GH-/- mice have significantly larger adipocytes and reduced fibrosis, neither of which occurred in perigonadal WAT, suggesting that GH has a more pronounced effect on subcutaneous WAT. Comparisons of GH-/- mice to previously published data on GHR-/- mice show a remarkably similar phenotype. Finally, we demonstrate that GH-/- mice are responsive to GH treatment, as shown by changes to serum IGF-1 levels; body length, weight, and composition; and insulin sensitivity. This study not only provides characterization of the first mouse line with targeted mutation of the GH gene but also indicates that GH gene disruption dramatically influences fibrosis of subcutaneous WAT.

Adipocyte-Specific GH Receptor-Null (AdGHRKO) Mice Have Enhanced Insulin Sensitivity With Reduced Liver Triglycerides.

Global GH receptor-null or knockout (GHRKO) mice have been extensively studied owing to their unique phenotype (dwarf and obese but remarkably insulin sensitive and long-lived). To better understand the influence of adipose tissue (AT) on the GHRKO phenotype, we previously generated fat-specific GHRKO (FaGHRKO) mice using the adipocyte protein-2 (aP2) promoter driving Cre expression. Unlike global GHRKO mice, FaGHRKO mice are larger than control mice and have an increase in white AT (WAT) mass and adipocyte size as well as an increase in brown AT mass. FaGHRKO mice also have an unexpected increase in IGF-1, decrease in adiponectin, no change in insulin sensitivity or liver triglyceride content, and a decreased lifespan. Extensive analysis of the aP2 promoter/enhancer by multiple laboratories has revealed expression in nonadipose tissues, confounding interpretation of results. In the current study, we used the adiponectin promoter/enhancer to drive Cre expression, which better targets mature adipocytes, and generated a new line of adipocyte-specific GHRKO (AdGHRKO) mice. AdGHRKO mice have an increase in adipocyte size and WAT depot mass in all depots except male perigonadal, a WAT accumulation pattern similar to FaGHRKO mice. Likewise, adiponectin levels and WAT fibrosis are decreased in both tissue-specific mouse lines. However, unlike FaGHRKO mice, AdGHRKO mice have no change in IGF-1 levels, improved glucose homeostasis, and reduced liver triglycerides. Thus, AdGHRKO mice should be valuable for future studies assessing the contribution of adipocyte GHR signaling in long-term health and lifespan.

Removal of growth hormone receptor (GHR) in muscle of male mice replicates some of the health benefits seen in global GHR-/- mice.

Global disruption of the GH receptor in mice (GHR-/-) produces a large and reproducible extension in lifespan. Since lack of GH action in muscle resulting in improved glucose homeostasis is potentially a mechanism by which GHR-/- mice are long-lived, and since no information on muscle-specific GHR disruption in females is available, we generated and characterized a line of muscle-specific GHR disrupted (MuGHRKO) mice. As expected, male MuGHRKO mice had improved fasting blood glucose, insulin, c-peptide, and glucose tolerance. In contrast, female MuGHRKO mice exhibited normal glucose, insulin, and glucose tolerance. Body weight was mildly but significantly altered in opposite directions in males (decreased) and females (increased) compared to controls. Grip strength and treadmill endurance were unchanged with advanced age in both sexes, suggesting that the direct action of GH on muscle has minimal effect on age-related musculoskeletal frailty. Longevity was unchanged in both sexes at Ohio University and significantly increased for males at University of Michigan. These data suggest that removal of GHR in muscle of male MuGHRKO mice replicates some of the health benefits seen in global GHR-/- mice including improvements to glucose homeostasis and smaller body weight in males, which may explain the trends observed in lifespan.

Liver-specific GH receptor gene-disrupted (LiGHRKO) mice have decreased endocrine IGF-I, increased local IGF-I, and altered body size, body composition, and adipokine profiles.

GH is an important regulator of body growth and composition as well as numerous other metabolic processes. In particular, liver plays a key role in the GH/IGF-I axis, because the majority of circulating "endocrine" IGF-I results from GH-stimulated liver IGF-I production. To develop a better understanding of the role of liver in the overall function of GH, we generated a strain of mice with liver-specific GH receptor (GHR) gene knockout (LiGHRKO mice). LiGHRKO mice had a 90% decrease in circulating IGF-I levels, a 300% increase in circulating GH, and significant changes in IGF binding protein (IGFBP)-1, IGFBP-2, IGFBP-3, IGFBP-5, and IGFBP-7. LiGHRKO mice were smaller than controls, with body length and body weight being significantly decreased in both sexes. Analysis of body composition over time revealed a pattern similar to those found in GH transgenic mice; that is, LiGHRKO mice had a higher percentage of body fat at early ages followed by lower percentage of body fat in adulthood. Local IGF-I mRNA levels were significantly increased in skeletal muscle and select adipose tissue depots. Grip strength was increased in LiGHRKO mice. Finally, circulating levels of leptin, resistin, and adiponectin were increased in LiGHRKO mice. In conclusion, LiGHRKO mice are smaller despite increased local mRNA expression of IGF-I in several tissues, suggesting that liver-derived IGF-I is indeed important for normal body growth. Furthermore, our data suggest that novel GH-dependent cross talk between liver and adipose is important for regulation of adipokines in vivo.

Direct and indirect effects of growth hormone receptor ablation on liver expression of xenobiotic metabolizing genes.

Detoxification of ingested xenobiotic chemicals, and of potentially toxic endogenous metabolites, is carried out largely through a series of enzymes synthesized in the liver, sometimes called "xenobiotic metabolizing enzymes" (XME). Expression of these XME is sexually dimorphic in rodents and humans, with many of the XME expressed at higher levels in females. This expression pattern is thought to be regulated, in part, by the sex differences in circadian growth hormone (GH) pulsatility. We have evaluated mRNA, in the liver, for 52 XME genes in male and female mice of four mutant stocks, with diminished levels of GH receptor (GHR) either globally (GKO), or in liver (LKO), fat (FKO), or muscle (MKO) tissue specifically. The data show complex, sex-specific changes. For some XME, the expression pattern is consistent with direct control of hepatic mRNA by GHR in the liver. In contrast, other XME show evidence for indirect pathways in which hepatic XME expression is altered by GH signals in fat or skeletal muscle. The effects of GHR-null mutations on glucose control, responses to dietary interventions, steroid metabolism, detoxification pathways, and lifespan may depend on a mixture of direct hepatic effects and cross talk between different GH-responsive tissues.

The role of GH in adipose tissue: lessons from adipose-specific GH receptor gene-disrupted mice.

GH receptor (GHR) gene-disrupted mice (GHR-/-) have provided countless discoveries as to the numerous actions of GH. Many of these discoveries highlight the importance of GH in adipose tissue. For example GHR-/- mice are insulin sensitive yet obese with preferential enlargement of the sc adipose depot. GHR-/- mice also have elevated levels of leptin, resistin, and adiponectin, compared with controls leading some to suggest that GH may negatively regulate certain adipokines. To help clarify the role that GH exerts specifically on adipose tissue in vivo, we selectively disrupted GHR in adipose tissue to produce Fat GHR Knockout (FaGHRKO) mice. Surprisingly, FaGHRKOs shared only a few characteristics with global GHR-/- mice. Like the GHR-/- mice, FaGHRKO mice are obese with increased total body fat and increased adipocyte size. However, FaGHRKO mice have increases in all adipose depots with no improvements in measures of glucose homeostasis. Furthermore, resistin and adiponectin levels in FaGHRKO mice are similar to controls (or slightly decreased) unlike the increased levels found in GHR-/- mice, suggesting that GH does not regulate these adipokines directly in adipose tissue in vivo. Other features of FaGHRKO mice include decreased levels of adipsin, a near-normal GH/IGF-1 axis, and minimal changes to a large assortment of circulating factors that were measured such as IGF-binding proteins. In conclusion, specific removal of GHR in adipose tissue is sufficient to increase adipose tissue and decrease circulating adipsin. However, removal of GHR in adipose tissue alone is not sufficient to increase levels of resistin or adiponectin and does not alter glucose metabolism.

A novel small molecule 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose mimics the antiplatelet actions of insulin.

BACKGROUND: We have shown that 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose (α-PGG), an orally effective hypoglycemic small molecule, binds to insulin receptors and activates insulin-mediated glucose transport. Insulin has been shown to bind to its receptors on platelets and inhibit platelet activation. In this study we tested our hypothesis that if insulin possesses anti-platelet properties then insulin mimetic small molecules should mimic antiplatelet actions of insulin. PRINCIPAL FINDINGS: Incubation of human platelets with insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1 and blocked ADP or collagen induced aggregation. Pre-treatment of platelets with α-PGG inhibited thrombin-induced release of P-selectin, secretion of ATP and aggregation. Addition of ADP or thrombin to platelets significantly decreased the basal cyclic AMP levels. Pre-incubation of platelets with α-PGG blocked ADP or thrombin induced decrease in platelet cyclic AMP levels but did not alter the basal or PGE(1) induced increase in cAMP levels. Addition of α-PGG to platelets blocked agonist induced rise in platelet cytosolic calcium and phosphorylation of Akt. Administration of α-PGG (20 mg kg(-1)) to wild type mice blocked ex vivo platelet aggregation induced by ADP or collagen. CONCLUSIONS: These data suggest that α-PGG inhibits platelet activation, at least in part, by inducing phosphorylation of insulin receptors leading to inhibition of agonist induced: (a) decrease in cyclic AMP; (b) rise in cytosolic calcium; and (c) phosphorylation of Akt. These findings taken together with our earlier reports that α-PGG mimics insulin signaling suggest that inhibition of platelet activation by α-PGG mimics antiplatelet actions of insulin.

Gene targeting implicates Cdc42 GTPase in GPVI and non-GPVI mediated platelet filopodia formation, secretion and aggregation.

BACKGROUND: Cdc42 and Rac1, members of the Rho family of small GTPases, play critical roles in actin cytoskeleton regulation. We have shown previously that Rac1 is involved in regulation of platelet secretion and aggregation. However, the role of Cdc42 in platelet activation remains controversial. This study was undertaken to better understand the role of Cdc42 in platelet activation. METHODOLOGY/PRINCIPAL FINDINGS: We utilized the Mx-cre;Cdc42(lox/lox) inducible mice with transient Cdc42 deletion to investigate the involvement of Cdc42 in platelet function. The Cdc42-deficient mice exhibited a significantly reduced platelet count than the matching Cdc42(+/+) mice. Platelets isolated from Cdc42(-/-), as compared to Cdc42(+/+), mice exhibited (a) diminished phosphorylation of PAK1/2, an effector molecule of Cdc42, (b) inhibition of filopodia formation on immobilized CRP or fibrinogen, (c) inhibition of CRP- or thrombin-induced secretion of ATP and release of P-selectin, (d) inhibition of CRP, collagen or thrombin induced platelet aggregation, and (e) minimal phosphorylation of Akt upon stimulation with CRP or thrombin. The bleeding times were significantly prolonged in Cdc42(-/-) mice compared with Cdc42(+/+) mice. CONCLUSION/SIGNIFICANCE: Our data demonstrate that Cdc42 is required for platelet filopodia formation, secretion and aggregation and therefore plays a critical role in platelet mediated hemostasis and thrombosis.

Endocrine parameters and phenotypes of the growth hormone receptor gene disrupted (GHR-/-) mouse.

Disruption of the GH receptor (GHR) gene eliminates GH-induced intracellular signaling and, thus, its biological actions. Therefore, the GHR gene disrupted mouse (GHR-/-) has been and is a valuable tool for helping to define various parameters of GH physiology. Since its creation in 1995, this mouse strain has been used by our laboratory and others for numerous studies ranging from growth to aging. Some of the most notable discoveries are their extreme insulin sensitivity in the presence of obesity. Also, the animals have an extended lifespan, which has generated a large number of investigations into the roles of GH and IGF-I in the aging process. This review summarizes the many results derived from the GHR-/- mice. We have attempted to present the findings in the context of current knowledge regarding GH action and, where applicable, to discuss how these mice compare to GH insensitivity syndrome in humans.