Pleckstrin homology domains interact with filamentous actin.

A fraction of Bruton's tyrosine kinase (Btk) co-localizes with actin fibers upon stimulation of mast cells via the high affinity IgE receptor (FcepsilonRI). In this study, a molecular basis of the Btk co-localization with actin fibers is presented. Btk and other Tec family tyrosine kinases have a pleckstrin homology (PH) domain at their N termini. The PH domain is a short peptide module frequently found in signal-transducing proteins and cytoskeletal proteins. Filamentous actin (F-actin) is shown to be a novel ligand for a subset of PH domains, including that of Btk. The actin-binding site was mapped to a 10-residue region of the N-terminal region of Btk. Basic residues in this short stretch are demonstrated to be involved in actin binding. Isolated PH domains induced actin filament bundle formation. Consistent with these observations, Btk binds F-actin in vitro and in vivo. Wild-type Btk protein is in part translocated to the cytoskeleton upon FcepsilonRI cross-linking, whereas Btk containing a mutated PH domain is not. Phosphatidylinositol 3,4, 5-trisphosphate-mediated membrane translocation of Btk was enhanced in cytochalasin D-pretreated, FcepsilonRI-stimulated mast cells. These data indicate that PH domain-mediated F-actin binding plays a role in Btk co-localization with actin filaments.

Galectin-3 inhibits granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven rat bone marrow cell proliferation and GM-CSF-induced gene transcription.

The expression of galectin-3 (formerly known as IgE-binding protein or Mac-2) in rat bone marrow (BM) was investigated by FACS, immunocytochemical and immunoblot analysis. The functional significance of rat recombinant galectin-3 on mouse recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven proliferation of macrophage progenitors and gene transcription was further examined. Immunocytochemical analysis of in situ BM sections demonstrated galectin-3 in myelopoietic cells and surrounding stroma, whereas erythropoietic and lymphopoietic environments essentially lacked galectin-3 expression. FACS analysis demonstrated that incubation of freshly isolated BMC with lactose, a competing ligand for galectin-3 binding to glycoconjugates, decreased binding of antigalectin antibodies to cells primarily expressing the myeloid antigen recognized by mAb His-54. Similarly, lectin-mediated binding of exogenous galectin-3 to myeloid lineage cells was also demonstrated. Immunoblot analysis of BM eluates demonstrated galectin-3 both in the extracellular matrix and in a lactose elutable form, bound to the surface of BMC. [3H]Thymidine incorporation studies on BMC cultured in the presence of galectin-3 demonstrated suppression of GM-CSF-induced proliferation by galectin-3. In addition, differential display analysis of immediate early gene expression in BMC cultured in the presence of galectin-3 revealed a 76.2% inhibition of GM-CSF-induced gene transcription by galectin-3 assessed by the number of PCR-fragments generated. Our data suggest a role for galectin-3 in the organization of myelopoietic compartments in rat BM and regulation of the action of growth factors on myelopoietic precursor cells.

A human lectin, galectin-3 (epsilon bp/Mac-2), stimulates superoxide production by neutrophils.

A family of soluble animal lectins, galectins, with beta-galactoside-binding activity, is gaining increased attention. One member of this family, galectin-3, has been previously designated by this group as epsilon bp, for its IgE-binding activity. On the basis of the saccharide specificity and other biochemical characteristics of epsilon bp, it is possible that this lectin could have an important extracellular modulatory role, functioning through recognition of critical cell surface glycoproteins on many cell types. We present evidence here that recombinant human epsilon bp activates human neutrophils in a dose-dependent manner as demonstrated by superoxide production. The observed activity is dependent on the lectin property of epsilon bp intrinsic to its carboxyl-terminal domain, as it could be inhibited effectively by lactose, a known saccharide ligand of epsilon bp. However, the amino-terminal domain is also necessary for the observed activity, as epsilon bp-C (the carboxyl-terminal domain fragment) is devoid of neutrophil-activating activity, even though it retains the carbohydrate-binding property. Affinity purification of lysates from cell surface-radio-iodinated neutrophils revealed two major protein bands of M(r) 115,000 and M(r) 180,000 that are recognized by epsilon bp and preliminary data suggested that one of these proteins is NCA-160, a human carcinoembryonic Ag-related glycoprotein. This study thus lends further support to our view of an extracellular function for epsilon bp and suggests that this protein has an important role in inflammation and host defense through modulating the function of neutrophils.

An endogenous lectin, galectin-3 (epsilon BP/Mac-2), potentiates IL-1 production by human monocytes.

Galectin-3 is a member of a growing family of beta-galactoside-binding animal lectins and previously designated as epsilon BP (IgE-binding protein) by this laboratory and as Mac-2, CBP35, L-34 and L-29 by other researchers. While possible intracellular functions have been proposed for galectin-3, existing data also suggest an extracellular modulatory role of this lectin. For example, epsilon BP/Mac-2 was found to be secreted by various cells and capable of activating mast cells, possibly through cross-linking of cell surface glycoproteins involved in cell activation. In this study, we showed that epsilon BP bound to human monocytes via its lectin function. Furthermore, we found that epsilon BP potentiated IL-1 production by monocytes in a manner that was inhibitable by the saccharide ligand of epsilon BP. The results further support a role of this lectin in potentiating activities of inflammatory cells and thereby amplifying inflammatory responses.

Activation of rat basophilic leukemia cells by epsilon BP, an IgE-binding endogenous lectin.

IgE-binding protein (epsilon BP) is a beta-galactoside-binding animal lectin identified by its affinity for IgE. We have reported that epsilon BP also binds the mast cell high-affinity IgE receptor (Fc epsilon RI), via lectin-carbohydrate interaction. We have now studied the physiological significance of epsilon BP-IgE-Fc epsilon RI interactions in mast cell activation using rat basophilic leukemia (RBL) cells as the model system. We report here that both unsensitized and IgE-sensitized RBL cells are activated upon exposure to epsilon BP-coated surfaces. Activation of RBL cells by the lectin epsilon BP can be significantly inhibited by appropriate saccharides. Exposure of RBL cells to epsilon BP-coated surfaces caused cell spreading similar to that caused from adherence to fibronectin-coated surfaces. However, epsilon BP by itself caused mediator release whereas fibronectin only potentiated antigen-mediated activation of RBL cells. Under appropriate conditions, epsilon BP, therefore, has the potential to activate mast cells culminating in augmentation of an inflammatory response.

The adhesive specificity of the soluble human lectin, IgE-binding protein, toward lipid-linked oligosaccharides. Presence of the blood group A, B, B-like, and H monosaccharides confers a binding activity to tetrasaccharide (lacto-N-tetraose and lacto-N-neotetraose) backbones.

The immunoglobulin E-binding protein, epsilon BP (also known as CBP35, Mac-2, L-34, and L-29), is a beta-galactoside-binding protein of approximately 30 kDa and a member of the animal lectin family termed S-type or S-Lac. Multiple biological activities have been attributed to this lectin such as mediation of IgE binding to the surface of Langerhans cells and activation of mast cells through binding to the high affinity IgE receptor. In order to better understand the cell-binding activity and the proposed role for epsilon BP as a biological response modifier, we have studied the specificity of binding of the radioiodinated epsilon BP to a series of lipid-linked, structurally defined oligosaccharide sequences of the lacto/neolacto family. The results show that the minimum lipid-linked oligosaccharides that can support epsilon BP binding are pentasaccharides of the lacto/neolacto series and that the lectin binds more strongly to oligosaccharides of this family that bear the blood group A, B, or B-like determinants than to those bearing blood group H. This preferential binding of epsilon BP is also manifest with whole cells, as erythrocytes of blood groups A and B are more strongly bound by epsilon BP than those of blood group O. Blood group Le(a) and Le(x) sequences are not bound by the lectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Obesity, diabetes, and neoplasia in yellow A(vy)/- mice: ectopic expression of the agouti gene.

The viable yellow A(vy) mutation results in a mottled yellow mouse that is obese, slightly larger than its nonyellow sibs, and more susceptible to tumor formation in those tissues sensitized by the strain genome. The mutation exhibits variable expressivity resulting in a continuum of coat color phenotypes, from clear yellow to pseudoagouti. The mouse agouti protein is a paracrine signaling molecule that induces hair follicle melanocytes to switch from the synthesis of black pigment to yellow pigment. Molecular cloning studies indicate that the obesity and growth effects of the A(vy) mutation result from ectopic expression of the normal agouti gene product. This review seeks to summarize the current state of knowledge regarding the obesity, stimulation of somatic growth, and enhancement of tumor formation caused by the A(vy) mutation, and to interpret these pleiotropic effects in terms of the normal function of the agouti protein.

Expression and function of an IgE-binding animal lectin (epsilon BP) in mast cells.

epsilon BP (IgE-binding protein) is a 31,000 M(r) protein originally identified in rat basophilic leukemia (RBL) cells. The protein is composed of two domains with the amino-terminal domain containing a highly conserved repetitive sequence and the carboxyl-terminal domain containing consensus sequences shared by other beta-galactoside-binding soluble lectins. The protein has wide tissue distribution, is found on cell surfaces and in extracellular milieu. By combined efforts from several research groups including ours a multifunctional nature of this lectin began to emerge. This review emphasizes the following characteristics of epsilon BP: (i) epsilon BP is secreted by cells such as macrophages; (ii) like many other lectins, epsilon BP functions at least bivalently; (iii) epsilon BP has specificity for distinct oligosaccharide structures that have a terminal galactose not masked by sialic acids; and (iv) in addition to binding IgE, epsilon BP binds to surfaces of various cell types via lectin-carbohydrate interaction. Importantly, epsilon BP binds to the IgE receptor on mast cells. We propose that epsilon BP can function as a modulatory protein on various cells by cross-linking critical cell surface glycoproteins. The proposed action of epsilon BP on mast cells is presented as a model.

Epsilon BP, a beta-galactoside-binding animal lectin, recognizes IgE receptor (Fc epsilon RI) and activates mast cells.

IgE-binding protein (epsilon BP) was originally identified in rat basophilic leukemia (RBL) cells by virtue of its affinity for IgE. epsilon BP is now known to be a beta-galactoside-binding lectin containing an S-type carbohydrate recognition domain. It is identical to a macrophage surface antigen, Mac-2, and lectins designated as CBP35, L-34, and RL-29, for which various functions have been suggested. Studies from other groups as well as ours have indicated that epsilon BP is secreted by cells such as macrophages and is present in extracellular fluids. We demonstrated previously that binding sites for epsilon BP are present on the surface of RBL cells. In this report, we show that epsilon BP binds to a small number of glycoprotein species on the surface of RBL cells. Significantly, one of these glycoproteins is the high-affinity IgE receptor (Fc epsilon RI). Preliminary studies showed that epsilon BP causes mediator release from RBL cells, possibly through cross-linking of Fc epsilon RI. The results suggest a function of epsilon BP as an activator of mast cells.

Surface expression of functional IgE binding protein, an endogenous lectin, on mast cells and macrophages.

IgE-binding protein (epsilon BP) is a galactoside-specific lectin containing an S-type carbohydrate-recognition domain. It was originally identified in rat basophilic leukemia cells and is now known to be identical to a macrophage surface Ag, Mac-2, and lectins designated as CBP 35/L-34/RL-29. It has also been related to a nonintegrin laminin-binding protein isolated from mouse macrophages. In this report we have shown the following: epsilon BP is present in variable amounts in several mast cell lines, and the surface expression of epsilon BP in these cell lines is quite variable and does not correlate with the total amount of epsilon BP in the cell. epsilon BP is displayed on the cell surface in a manner that is reversible by lactose, most likely through attachment to yet unidentified glycoconjugates. The putative epsilon BP binding sites on the cell surface can be readily demonstrated by using radiolabeled epsilon BP, and the sites are present in comparable amounts in various cell lines. Expression of epsilon BP on the cell surface can be regulated; the most notable example is the upregulation of surface epsilon BP on RBL cells activated through the high-affinity IgE receptor by IgE immune complexes. Cell-surface epsilon BP is functional as measured by its ability to promote adhesion of trypsinized rabbit erythrocytes to mast cells and macrophages. On the basis of these results and reported properties of related lectins, we propose that the lectin represented by epsilon BP is a new class of cell-adhesion protein.

An intestinal galactose-specific lectin mediates the binding of murine IgE to mouse intestinal epithelial cells.

A number of lactose-binding lectins have recently been identified in the rat and mouse intestine, one of which corresponds to the C-terminal domain of IgE-binding proteins, originally identified in rat basophilic leukemia (RBL) cells and mouse 3T3 fibroblasts. In the present report, we describe the affinity purification of a rat intestinal lactose-specific lectin which binds murine IgE antibodies. This binding most likely occurs via the immunoglobulin carbohydrate chains, as it is inhibited by lactose. This intestinal lectin molecule is also immunologically related to the previously described IgE-binding protein (epsilon BP) isolated from RBL cells, since it is recognized by antibodies raised against recombinant epsilon BP. This intestinal form of epsilon BP has a molecular mass of 17.5 kDa, which is much lower than that of its RBL cell analogue (31 kDa). The attachment of IgE to the mouse intestinal epithelium was demonstrated by immunohistochemistry, along with the presence of a corresponding mouse intestinal epsilon BP. The carbohydrate-dependent nature of this attachment was established by demonstrating that IgE binding to mouse epithelium was specifically abolished by lactose (4 mM) and by a blood-group-A-active tetrasaccharide (0.2 mM), but not by mannose (10 mM). Finally, the association of IgE with the mouse intestinal epithelium was prevented by competition with the purified IgE-binding lectin isolated from rat intestine. Although the physiological function of this intestinal protein is still unknown, the finding that IgE binds to a lectin in the intestinal epithelium pinpoints a possible novel mechanism for the regulation of IgE-mediated disorders, such as food allergy.

Expression of biologically active recombinant rat IgE-binding protein in Escherichia coli.

IgE-binding protein (epsilon BP) is a protein which has affinity for IgE and was originally identified in rat basophilic leukemia (RBL) cells. Subsequently, it was found to be the rat homolog of CBP35, a murine beta-galactoside-specific lectin. This protein is also designated as L-34 and RL-29 and studied independently by several laboratories. More recently, CBP35 (epsilon BP) was found to be equivalent to Mac-2, a surface marker on activated macrophages. Using rat epsilon BP cDNA, we have succeeded in expressing recombinant epsilon BP in Escherichia coli. Milligram quantities of homogeneous epsilon BP could be obtained from bacterial lysate in a one-step affinity purification procedure utilizing lactosyl-Sepharose 4B and elution with a lactose gradient. The recombinant epsilon BP (r epsilon BP) binds mouse IgE and retains reactivity to anti-peptide antibodies specific for a sequence within rat epsilon BP. The purified r epsilon BP exhibits binding activity to various saccharides, with affinity for N-acetyllactosamine greater than thiodigalactoside greater than lactose much greater than D-galactose greater than L-arabinose, an order identical to that exhibited by native epsilon BP isolated from RBL cells. The recombinant lectin displayed hemagglutination activity when tested with rabbit erythrocytes. Although epsilon BP shares sequence homology to other lectins containing S-type (thiol-dependent) carbohydrate-recognition domains, r epsilon BP is resistant to air oxidation and does not require reducing agents for maintaining its activity. Furthermore, the single cysteine residue appears to be unexposed and can be alkylated only when the protein is denatured in 5.6 M guanidinium hydrochloride. The availability of a source for a large quantity of epsilon BP should facilitate the analysis of biological function(s) and structure-activity relationships of this lectin.

Diabetogenic response to streptozotocin varies among obese yellow and among lean agouti (BALB/c x VY)F1 hybrid mice.

To test the hypothesis that the elevated insulin levels in obese neoplasia-susceptible yellow Avy/- mice might be a major factor stimulating tumor formation, it is necessary to use normoinsulinemic yellow mice. Although our attempt to obtain normoinsulinemic, euglycemic mice by streptozotocin treatment was unsuccessful, we did observe significant differences in the responsiveness to this treatment among mice of identical genotype. These differences were observed among female yellow Avy/A and agouti A/a (BALB/c x VY)F1 hybrid mice in the responses of body weight gain, plasma glucose, and plasma insulin levels to a single intraperitoneal injection of either 150 or 200 mg/kg streptozotocin (STZ) at 4 weeks of age followed by a 22-week observation period. Among animals treated with the high streptozotocin dose, 80% of the yellow mice gained almost no weight and became grossly hyperglycemic and hypoinsulinemic; however, only 55% of the agouti mice exhibited such a strong response. In the low dose group, 25% of the yellow mice responded with reduced body weight gain, decreased insulin, and elevated glucose levels whereas none of the agouti mice exhibited such responses. More pancreatic islet tissue mass was present in the untreated yellow control mice than among the comparable agouti mice by the end of the study. In both streptozotocin dose groups and in both genotypes, islet tissue mass was reduced to a much greater extent in the more responsive mice than in the less responsive mice. There appeared to be no correlation between islet tissue mass and insulin level. The phenotypic variation in responsiveness to an exogenous agent among test animals of a single inbred or F1 hybrid genotype reported here is not unique to this F1 hybrid since it is seen in most chronic bioassays when relatively low levels of agent are used.

Amino-terminal peptide of growth hormone enhances insulin action in normal rats.

Earlier studies demonstrated that the 20K-dalton variant of human GH (hGH), which differs from hGH by deletion of the amino acid residues 32-46, has decreased insulin-like activity. The current study assessed whether a peptide representing this deleted region could enhance insulin-stimulated glucose uptake in the intact rat and if this effect was localized in liver and/or muscle. Peptides hGH-(32-46) and rat GH-(32-45) were used in these studies. Assessment of the action of a peptide was done by determining its effect on the steady state serum glucose (SSSG) concentration during an insulin suppression test. Glucose was infused alone and with two rates of infusion of insulin. The data indicated that neither of the GH peptides affected SSSG in the absence of exogenously administered insulin or at low (40 microU/ml) serum levels of insulin, but when serum insulin was increased to 77 microU/ml, a significant (P less than 0.05) decrease in SSSG was produced by the peptides. In subsequent studies isolated liver and hind limb skeletal muscle were perfused with a solution of hGH-(32-46). Basal glucose release from the liver was suppressed by both hGH-(32-46) and insulin alone, and this decrease was not enhanced by combining insulin with hGH-(32-46). Glucose uptake by skeletal muscle, expressed as a metabolic clearance constant (k), was enhanced by infusion of insulin and further increased with added peptide. Basal uptake was 6.67 microliter/min.g muscle; uptake was 8.17 microliter/min.g (P less than 0.01) after addition of 128 microU/ml insulin. This increased still farther to 9.24 microliter/min.g (P less than 0.05) when peptide was administered with insulin. These findings suggest that GH peptides independently suppress glucose outflow from the liver and potentiate insulin action by facilitating glucose uptake by peripheral tissues.

Increased sensitivity of adipose tissue to insulin after in vivo treatment of yellow Avy/A obese mice with amino-terminal peptides of human growth hormone.

Treatment of obese yellow Avy/A mice with the human GH (hGH) peptide hGH-(1-43) enhanced the in vitro sensitivity of their adipose tissue to insulin. Insulin-stimulated glucose oxidation, as determined by measurement of 14CO2 production, was enhanced 106% after administration of hGH-(1-43) at a dosage of 1 microgram/day for 3 days. A significant increase in CO2 production was detected with as little as 100 ng peptide/day for 3 days. A single injection of 10 micrograms increased sensitivity to insulin 2-5 times. This enhancing effect of insulin action could not be seen in lean agouti A/a animals nor could it be demonstrated by in vitro addition of hGH-(1-43) to adipose tissue. Synthetic hGH-(1-43) was used for these studies, but initial physiological work was done with peptide isolated from pituitary glands. At equimolar doses, intact hGH, a trypsin digest of either hGH or BSA, carbidomethyl cysteine-hGH-(146-191), and hGH-(32-46) were inactive. Carbidomethyl cysteine-hGH-(1-139) and hGH-(1-15) showed the enhancing property, but were only about 10% as active as hGH-(1-43). HGH-(1-43) did not increase serum insulin concentrations in the obese mice. We conclude that when administered in vivo to obese mice, hGH-(1-43) enhances the sensitivity of adipose tissue to the action of insulin, an indication that the peptide may play a role in carbohydrate metabolism.

Comparison of the acute metabolic effects of 22,000-dalton and 20,000-dalton growth hormone in human subjects.

The acute metabolic effects of 20,000-dalton human growth hormone (hGH20K) in man have not previously been tested. We compared changes in concentrations of free fatty acids (FFA), glucose, and insulin in nine growth hormone deficient children following injection of 22,000-dalton intact human growth hormone (hGH22K) and the smaller variant, hGH20K. There was a significant decline (37%) in the mean FFA concentration from baseline to 1/2 hour post-injection and from baseline to 1 hour post-injection (36%) in the children given hGH22K, but no such decline was seen after injection of hGH20K. No significant differences in mean insulin or glucose concentrations were noted between the two treatment groups, and glucose and insulin concentrations did not acutely change after injection of either hormone. The results of this study indicate that hGH20K has a diminished activity for suppression of FFA as compared to hGH22K. This suggests that GH residues 32-46, missing in hGH20K, constitute all or part of the region of hGH22K producing this response, or that the different primary structures of the two hormones result in tertiary structural differences and altered biological activity.

Differential responses of yellow Avy/A and agouti A/a (BALB/c X VY) F1 hybrid mice to the same diets: glucose tolerance, weight gain, and adipocyte cellularity.

Differences in weight gain, efficiency of food utilization, glucose tolerance, insulin levels, and adipocyte cellularity were measured when three different diets were fed to lean agouti and obese yellow mice. Sets of adult and weanling agouti (A/a) and yellow (Avy/A) (BALB/c X VY) F1 hybrid mice were fed high-sucrose (HS), 10 percent fat, or regular lab chow (control). Some mice received the diets only after 12 weeks of eating lab chow (adult-fed); others ate the diets from the time of weaning. Since responses of both age groups were similar, only the data from the adult-fed groups are presented. The HS and 10 percent fat diets increased the body weight gain in both A/a and Avy/A adult mice more than the control diet; the HS diet was utilized more efficiently in the lean agouti females causing a slightly higher weight gain, whereas the 10 percent fat and HS diets were used with the same efficiency by the yellow mice. Short-term feeding (3-5 weeks) with the HS and 10 percent fat diets decreased the glucose tolerance of adult yellow but not of agouti mice. The pancreatic insulin response to a glucose load was higher in all mice fed the HS diet, whereas this response was blunted in those on the 10 percent fat diet. In agouti mice the HS and 10 percent diets increased the mean cell diameter of the parametrial adipocytes and deteriorated the glucose oxidation rate in response to insulin compared to the control diet. In the yellow littermates, on the other hand, the test diets decreased the mean cell diameter and also impaired insulin sensitivity of the adipocytes. The decrease of the mean adipocyte size was probably due to an increased number of small cells.

In vitro insulin-like actions of human growth hormone: a study with an insulin antibody.

When polyclonal insulin antibodies were preincubated with either adipose tissue from hypophysectomized rats or adipocytes from normal rats, human growth hormone failed to stimulate glucose oxidation. Removal of insulin from adipocytes through incubation with pyruvate at pH 7.0, followed by washing three times, also abolished subsequent in vitro insulin-like action of hGH. Administration of the same insulin antibody to hypophysectomized rats 30 minutes prior to injection of hGH did not inhibit the insulin-like activity of the hGH as measured by its ability to decrease serum glucose and non-esterified fatty acid levels. It is concluded that the in vitro promotion of glucose oxidation by hGH requires insulin. Because of the uncertainty of complete removal of insulin in intact animals, such a conclusion cannot be made regarding in vivo insulin-like action of hGH.

Glycosylation abolishes an in vitro insulin-like action of prolactin.

Ovine prolactin stimulated 14C-CO2 production from labeled glucose in adipose tissue of hypophysectomized rats in vitro, an insulin-like activity. Glycosylation of the hormone by attachment of a carbohydrate unit at asparagine31 abolished this in vitro insulin-like action. However, neither nonglycosylated nor glycosylated prolactin exhibited in vivo insulin-like action, as they did not lower serum glucose or non-esterified fatty acids in fasted hypophysectomized rats. Hindrance of receptor binding by the carbohydrate unit may account for the absence of in vitro insulin-like activity in glycosylated prolactin, but the dichotomy between in vivo and in vitro insulin-like actions for prolactin remains obscure.

Impairment of glucose tolerance in yellow (Avy/A) (BALB/c X VY) F-1 hybrid mice by hyperglycemic peptide(s) from human pituitary glands.

A fraction of human pituitary extract containing low molecular weight peptide(s) was found to impair glucose tolerance in obese yellow (Avy/A) (BALB/c X VY) F-1 hybrid female mice. In these mice, purified human GH was not hyperglycemic. Glucose tolerance of untreated yellow mice was somewhat less impaired than that of untreated ob/ob mice, and priming with dexamethasone was not required to elicit impairment of glucose tolerance. The lean agouti (A/a) littermates of the yellow mice did not respond to the hyperglycemic peptide(s), suggesting that an insulin-resistant animal is required for assay of the pituitary hyperglycemic peptide(s). Plasma insulin levels of the obese yellow mice treated with the hyperglycemic peptide(s) were significantly decreased compared to those of saline-treated controls. No decrease in plasma insulin was observed in the similarly treated lean agouti mice. The study demonstrated that the yellow Avy/A (BALB/c X VY) F-1 hybrid mouse is a suitable animal for investigation of the physiological mechanism of action of the pituitary hyperglycemic peptide(s).