The ob/ob mouse responds predictably to chronic treatment with large doses of pituitary GH with marked hyperglycemia and decreased glucose tolerance. The purpose of the present study was to characterize the metabolic alterations produced by GH that lead to this diabetogenic response in the ob/ob mouse in order to determine whether this animal might serve as a useful model for the study of the cellular mechanisms involved in the diabetogenic action of GH. Female ob/ob mice were treated sc for 3 days with either saline or 200 micrograms/day S-carboxymethylated human GH (RCM-hGH), a diabetogenic GH derivative lacking significant growth-promoting or insulin-like activities. Six hours before the start of the experiment, the animals were given a sc injection of 2 micrograms dexamethasone and deprived of food. RCM-hGH treatment produced marked increases in fasting blood glucose and plasma insulin concentrations, but had no effect on plasma glucagon or serum insulin-like growth factor I levels. It had no effect on liver glycogen level or in vitro hepatic glucose production in the absence or presence of pyruvate and lactate added to the incubation medium. By contrast, the in vitro stimulatory effects of insulin on [14C] glucose oxidation by isolated soleus muscle or segments of parametrial fat were greatly attenuated by RCM-hGH treatment, without changes in rates of basal glucose oxidation. This change in peripheral tissue responsiveness to insulin does not appear to involve glucose transport, since the in vitro stimulation by insulin of 3-O-[14C]methylglucose transport into isolated diaphragm muscle was not altered by RCM-hGH treatment. Moreover, the RCM-hGH-induced reduction in adipose tissue responsiveness to insulin does not appear to be mediated by a reduction in insulin binding, since [125I]iodoinsulin binding to adipocytes isolated from RCM-hGH-treated mice was similar to that to cells from saline-treated animals. Interestingly, the reduction in responsiveness to insulin seen with segments of adipose tissue from RCM-hGH-treated animals was not found with isolated adipocytes prepared from such tissue by collagenase digestion. These results suggest that the hyperglycemia and glucose intolerance produced in ob/ob mice by chronic GH treatment result primarily from increased peripheral tissue insulin resistance. Therefore, the ob/ob mouse provides a useful model to elucidate the cellular mechanism(s) of this aspect of the diabetogenic action of GH.

The anterior pituitary gland produces a 20-kilodalton (kDa) variant of human growth hormone (hGH) that differs from the predominant 22-kDa form of hGH in that amino acid residues 32-46 are deleted. Previous work has suggested that the 20-kDa variant possesses the full growth-promoting and lactogenic activities of 22-kDa hGH but lacks its intrinsic diabetogenic and insulin-like activities. In the present study, recombinant DNA techniques were used to prepare biosynthetic 20-kDa hGH, and some of the biological properties of the purified hGH variant were examined. The biosynthetic 20-kDa hGH variant was found to share the propensity for aggregation exhibited by its native counterpart. Moreover, like the native variant, biosynthetic 20-kDa hGH possessed full growth-promoting activity in the weight gain test in hypophysectomized rats. However, contrary to previous work suggesting that native 20-kDa hGH lacks diabetogenic and insulin-like activities, biosynthetic 20-kDa hGH was found to have substantial diabetogenic activity when administered chronically to ob/ob mice and to possess approximately 20% the in vitro insulin-like activity of biosynthetic 22-kDa hGH on isolated epididymal adipose tissue of hypophysectomized rats. The diabetogenic and insulin-like activities of biosynthetic 20-kDa hGH cannot be ascribed to contamination of the hormone preparation with the 22-kDa form of hGH or with other diabetogenic or insulin-like pituitary peptides. Therefore, the results strongly suggest that diabetogenic and insulin-like activities are also intrinsic properties of the 20-kDa variant of hGH.A 20-kilodalton variant of human growth hormone (20-kDa hGH) has been isolated from human pituitary glands (1) and has been found to differ from the predominant 22-kilodalton form of hGH (22-kDa hGH) in that residues 32-46 are deleted (2, 3). The variant is produced in the human pituitary, because the nucleotide sequence encoding residues 32-46 is sometimes excised during the processing of hGH pre-mRNA to hGH mRNA (4). As a consequence, approximately 5-10%1 of the hGH present in the human pituitary is 20-kDa hGH (1). Physicochemical studies of 20-kDa hGH (3) suggest that its conformation is similar but not identical to that of 22-kDa hGH.The 20-kDa variant of hGH has been reported (1, 5) to have growth-promoting activity in the hypophysectomized rat equivalent to that of 22-kDa hGH. Moreover, its lactogenic activity in the pigeon crop sac assay equals that of 22-kDa hGH (1). On the other hand, Lewis et al. (5) reported that 20-kDa hGH lacked diabetogenic activity, in that it failed to produce hyperglycemia and glucose intolerance in dogs, when administered 10 hr prior to a glucose tolerance test. Also, this same group reported (6) that 20-kDa hGH lacked the acute insulin-like property of 22-kDa hGH, since the 20-kDa hGH variant failed to produce hypoglycemia and increase plasma free fatty acids when administered to hypophysectomized rats and did not increase glucose uptake or oxidation to CO2 by isolated rat adipose tissue wh...

Lipid biosynthesis in cultured mammary tissues from mice during midpregnancy was maximally stimulated by the combined actions of PRL, insulin, and a glucocorticoid. The minimal concentration of cortisol that was consistently permissive for the action of PRL on lipid synthesis was 0.1 microgram/ml (2.76 X 10(-7) M). The PRL effect began after 6-8 h of exposure to PRL. The response to PRL was essentially all or none, with 25 ng/ml sufficient for maximal stimulation. Specificity of the PRL effect is suggested by the observation that 1 microgram/ml bovine GH was without effect in cultured mammary tissues. Additionally, PRL had no effect on lipid metabolism in explants of ovarian fat pad, suggesting that the PRL effect in the mammary gland is not a generalized effect on fat cells. The action of PRL in the mammary gland requires both ongoing RNA and protein syntheses, since both actinomycin D and puromycin abolished its effect. The early action of PRL on lipid biosynthesis was specific for the formation of triglycerides, but not other lipid classes studied.

GH has acute stimulatory effects on amino acid transport and protein synthesis in a variety of tissues, but it has not been established whether these effects are expressions of the growth-promoting property of GH or of its separate insulin-like action. The 20,000-dalton structural variant of human GH (20K hGH) has been shown to have a high ratio of growth-promoting to insulin-like activity compared to native hGH (22K hGH), suggesting that it could be used as a tool to address the above question. Therefore, experiments were conducted to compare the relative abilities of native 22K hGH and 20K hGH, when added in vitro, to stimulate amino acid transport and protein synthesis in the isolated diaphragm of the female hypophysectomized rat. Paired intact hemidiaphragms were preincubated for 1 h in the absence or presence of various concentrations of 22K or 20K hGH. Then, 3-O-[14C]methylglucose was added to the medium to measure sugar transport as a test of insulin-like activity, and either alpha-[3H]aminoisobutyric acid acid or [3H] phenylalanine was also added to measure amino acid transport or protein synthesis, respectively, during a final hour of incubation. When the responses to the various concentrations of 22K and 20K were compared, 20K hGH was only about 20% as effective as 22K in stimulating 3-O-methylglucose transport, reflecting its markedly attenuated insulin-like activity on the diaphragm. Similarly, 20K hGH was only 20% as effective as 22K hGH in stimulating alpha-aminoisobutyric acid transport and phenylalanine incorporation into protein in the same muscles. Therefore, these findings support the idea that the rapid stimulatory effects of GH on amino acid transport and protein synthesis are expressions of the insulin-like action of GH and are not components of the response of target cells to its growth-promoting action.