The characteristics of hGH binding to the liver macrophages

Macrophages isolated from female rat liver as well as hepatocytes bind 125I-hGH. This study compares the effect of sex of the rat, hypophysectomy (hypox) and preincubation of the cells with oPrl on the binding of 125I-hGH to the cells. The percent of 125I-hGH to the hepatocytes was decreased in cells from hypox female and male rats, and hepatocytes preincubated with oPrl to 0.43, 0.21 and 0.39, respectively, of that observed in hepatocytes from normal female rats. In the hepatocytes from normal female, hypox female, and male rats, hGH was the most effective competitor for 125I-hGH binding with an ID50 of 0.73-0.99 nM. The concentration of oPrl, bGH and rGH that produced half-maximal inhibition (ID50) of 125I-hGH binding to hepatocytes from female rat liver was 6.3, 100, and 420 nM respectively. In hepatocytes from male and hypox female rats, and hepatocytes preincubated with oPrl, the ID50 for bGH and rGH varied from 2.1 to 15.9 nM. The percent of 125I-hGH bound by the macrophages from hypox female and male rats, and macrophages preincubated with oPrl was 0.06, 0.15 and 0.18, respectively, of that bound by macrophages from normal female rat liver. In contrast to hGH binding to the hepatocytes, the ID50 for hGH was 6 to 180-fold greater in macrophages from hypox female and male rats, and macrophages preincubated with oPrl compared to that observed in macrophages from normal female rats, Rat GH was the most effective competitor for 125I-hGH binding in the macrophages from the hypox female and male rat liver with ID50 of 5.5 and 85 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased growth hormone binding to liver membranes of obese Zucker rats

Developmental changes in hepatic growth hormone binding sites were examined in the genetically obese male fa/fa rats and in the lean littermates. At 16 days, fa/fa pups are normoinsulinemic; the specific binding of 125I-hGH to liver membranes is comparable in the two genotypes. At 4 weeks and later on, plasma membranes and Golgi fractions of male obese Zucker rats have more GH binding sites than lean littermates. The GH pituitary content is comparable in the two genotypes from 2 to 8 weeks and in 14-week-old fa/fa rats it is half that in lean animals. In the two genotypes plasma IGFI dramatically increases during puberty. At 4 weeks, plasma IGFI level is significantly higher in fa/fa rats than in lean littermates. In this model of genetic obesity, an increased GH binding to liver membranes is observed after the third week of life, shortly after the onset of hyperinsulinemia in the fa/fa rat.     

In vitro insulin-like actions of the growth factor from the tapeworm, Spirometra mansonoides

In vitro actions of purified plerocercoid growth factor (PGF) were compared with those of insulin and human growth hormone (hGH) in adipose tissue from normal male rats. Insulin-like effects were measured by the ability of PGF, insulin, or hGH to stimulate oxidation of [U-14C]glucose to 14CO2, to stimulate lipogenesis, and to inhibit epinephrine-induced lipolysis. PGF and insulin stimulated significant increases in glucose oxidation and lipogenesis in adipose tissue that had not been preincubated as well as in tissue that had been preincubated. hGH stimulated insulin-like effects only in tissue that had been preincubated for 3 hr. Insulin, hGH, and PGF inhibited epinephrine-induced lipolysis of preincubated (3 hr) adipose tissue. hGH produced a dramatic lipolytic response in tissue freshly removed from normal rats but no dose of PGF was lipolytic. PGF did not displace 125I-insulin from its receptors on adipocytes but did competitively inhibit 125I-hGH binding to adipocytes. These results suggest that PGF has direct insulin-like actions which are initiated by binding a GH receptor, but PGF had no anti-insulin action and the insulin-like activity of PGF was unaffected by refractoriness of adipose tissue to GH.     

Species variation in the binding of hGH to hepatic membranes

The binding of 125I-labeled human growth hormone (hGH) to liver membranes from several different species was studied to determine the lactogenic or somatotropic hormone nature of the receptors. Liver membranes from several species of the class of Mammalia bound significant quantities of 125I-hGH. Goat, sheep, rat, mouse, and rabbit liver membranes exhibited the highest binding with cow, pig, human, and hamster liver membranes exhibiting severalfold less binding. The binding of the dog and cat liver membranes exhibited relatively high nonspecific binding. Fish and chicken liver membranes did not bind appreciable quantities of 125I-hGH. In all species except for dog and cat in which 125I-hGH bound to the membranes, hGH was the most effective competitor for binding. The mean ID50 for hGH and all membranes was 2.4 X 10(-9) M. Human liver membranes exhibited the smallest ID50, 4.9 X 10(-10) M. In sheep liver membranes, bovine growth hormone (bGH) was equipotent to hGH in competing for 125I-hGH binding. bGH also demonstrated significant competition for 125I-hGH binding in pig and cow membranes. Ovine prolactin (oPrl) exhibited significant competition for 125I-hGH only in rodent membranes. The ID50 for oPrl was 3- to 10-fold greater than for hGH in the rat, hamster, and mouse liver membranes. The ID50 for oPrl in the sheep liver membranes was 13-fold greater than that of hGH. We conclude the following: (1) There appears to be a species specificity of hGH binding that may be phylogenetically significant and may result from variations in the structure of the hormone or the receptor. (2) The competitive binding properties of hGH are fairly consistent within phylogenetic orders. (3) The simple designation of lactogenic or somatotropic for hormones and receptors is insufficient to characterize the binding properties of this group of hormones.     

Induction of specific human growth hormone binding sites in rat liver by human growth hormone.

125I-Labeled hGH was bound to liver plasma membranes which were obtained from female rats. The binding was displaced by hGH, hPRL, bPRL, rPRL and bGH but not by rGH. This result indicated that hGH was bound to lactogenic binding sites in rat livers. After hypophysectomy, the binding was markedly decreased. Treatment of hypophysectomized rats with hGH (80 micrograms/day) for 10 days increased the binding sites for hGH. These binding sites were different from those found in normal female rat livers because of their high affinity and specificity for hGH. These results indicate that hGH induces specific binding sites for hGH in rat livers.     

Identification of somatogenic binding sites in liver microsomes from normal mice and transgenic mice expressing human growth hormone gene.

Somatogenic binding sites were detected and characterized in microsomal preparations from livers of normal mice and mice expressing metallothionein-I/hGH (mMT/hGH) hybrid gene, using 125I-labelled bovine or human GH, or a photoreactive derivative of hGH (125I-AP-hGH1). Specific binding of 125I-bGH was detected in liver microsomes from both normal and transgenic mice with an apparent Kd of 2 nM. 125I-hGH was partially displaced by bGH. 125I-AP-hGH1 was covalently bound to the microsomal preparations, and bGH prevented the formation of the 130 kDa species with no appreciable effect on 63 kDa and 70 kDa lactogenic complexes.     

Subdiaphragmatic vagal deafferentation affects body weight gain and glucose metabolism in obese male Zucker (fa/fa) rats

We investigated the effect of subdiaphragmatic vagal deafferentation (SDA) on food intake, body weight gain, and metabolism in obese (fa/fa) and lean (Fa/?) Zucker rats. Before and after recovery from surgery, food intake and body weight gain were recorded, and plasma glucose and insulin were measured in tail-prick blood samples. After implantation of a jugular vein catheter, an intravenous glucose tolerance test (IVGTT) was performed, followed by minimal modeling to estimate the insulin sensitivity index. Food intake relative to metabolic body weight (g/kg(0.75)) and daily body weight gain after surgery were lower (P < 0.05) in SDA than in sham obese but not lean rats. Before surgery, plasma glucose and insulin concentrations were lower (P < 0.05) in lean than in obese rats but did not differ between surgical groups within both genotypes. Four weeks after surgery, plasma glucose and insulin were still similar in SDA and sham lean rats but lower (P < 0.05) in SDA than in sham obese rats. IVGTT revealed a downward shift of the plasma insulin profile by SDA in obese but not lean rats, whereas the plasma glucose profile was unaffected. SDA decreased (P < 0.05) area under the curve for insulin but not glucose in obese rats. The insulin sensitivity index was higher in lean than in obese rats but was not affected by SDA in both genotypes. These results suggest that elimination of vagal afferent signals from the upper gut reduces food intake and body weight gain without affecting the insulin sensitivity index measured by minimal modeling in obese Zucker rats.     

Isolated adipocytes from growth hormone-treated obese (ob/ob) mice exhibit insulin resistance

The genetically obese (ob/ob) mouse is a useful model for the study of the diabetogenic action of growth hormone (GH), because treatment of these animals with GH results in decreased responsiveness of their adipose tissue to insulin in vitro. Studies of the mechanisms involved in GH-induced insulin resistance using isolated adipocytes of ob/ob mice have not been possible, however, because of their extreme fragility and the lack of an adequate system for the maintenance of these cells. This study describes a new method for the isolation of ob/ob mouse adipocytes. The isolated cells are stable, viable and metabolically responsive to insulin. In addition, these adipocytes have been maintained in primary culture, in serum-free medium, for up to 3 days. During culture, the cells exhibit large increases in 125I-hGH binding (10-20-fold) and porcine 125I-insulin binding (5-10-fold). The induction of insulin resistance by GH has also been demonstrated in these freshly isolated ob/ob mouse adipocytes. The studies to date indicate that the ob/ob mouse adipocyte system should provide a useful model for detailed studies of the cellular and molecular mechanisms of GH induced insulin resistance.     

High affinity GDP binding sites on brown adipose tissue mitochondria of genetically obese ‘fa/fa’ rats

The number of high affinity [³H]GDP binding sites in brown adipose tissue mitochondria is normal in obese ( f a / f a ) rats in contrast to the reduced number of low affinity GDP binding sites. Adrenalectomy corrected the loss of low affinity binding sites in fa/fa rats but had no effect on the number of high affinity sites in either lean or obese rats. Equilibrium dialysis was used to show the presence of both high and low affinity binding sites on the purified 32 kdalton protein.     

Insulin binding in the hypothalamus of lean and genetically obese Zucker rats

Recent reports have suggested that the obesity and hyperphagia of the genetically obese Zucker rat may be related to defective insulin action or binding in the hypothalamus. We used quantitative autoradiography to determine if insulin binding is altered in specific hypothalamic nuclei associated with food intake. Insulin binding was measured in the arcuate (ARC), dorsomedial (DMN), and ventromedial (VMN) hypothalamic nuclei of 3–4-month-old lean (Fa/Fa) and genetically obese (fa/fa) Zucker rats. A consistently reproducible 15% increase in the total specific binding of 0.1 nM [¹²⁵I]-insulin was found in the ARC of the obese genotype. A slight increase in insulin binding in the DMN was also found. No difference in specific insulin binding was found between genotypes in the VMN. Nonlinear least squares analysis of competitive binding studies showed that the K_d of the ARC insulin binding site was 33% higher in the lean rats than in the obese rats, indicating an increased affinity for insulin. No difference in site number (B_max) was found in the ARC, DMN or VMN, and no evidence was found for reduced insulin binding in the hypothalamus of the obese (fa/fa) genotype. The results suggest that hyperphagia and obesity of the obese (fa/fa) Zucker rat genotype may be associated with increased insulin binding in the arcuate nucleus.     

IGF-I- and IGFBP-3-expression in cultured human preadipocytes and adipocytes.

The expression and secretion of IGF-I and IGFBP-3 were investigated in cultured human preadipocytes and in in vitro differentiated adipocytes derived from human subcutaneous adipose tissue under chemically defined culture conditions. Human preadipocytes expressed mRNAs for IGF-I and IGFBP-3 and secreted the corresponding proteins into the culture medium as measured by sensitive radioimmunoassays. In human adipocytes; specific mRNA-expression was comparable to that found in preadipocytes, but IGF-I secretion was increased 10-fold (3.87 +/- 0.69 vs. 0.41 +/- 0.11 ng/ml/10(6) cells/48 hrs, p < 0.05) and IGFBP-3 secretion 2.5-fold (7.34+/-1.15 vs. 3.27+/-0.38 ng/ml/10(6) cells/48 hrs, p<0.05) in the presence of adipogenic medium probably resulting in an increase of unbound IGF-I. Under serum-free, chemically defined conditions human growth hormone (hGH) and insulin were found to be positive regulators and cortisol was found to be a negative regulator of IGF-I and IGFBP-3 secretion in preadipocytes. In cultured human adipocytes, hGH showed no effect on IGF-I and IGFBP-3 secretion, whereas insulin stimulated and cortisol inhibited the secretion of both proteins. We conclude that IGF-I and IGFBP-3 may not only exert their actions in human adipose tissue via circulation, but also in an auto/paracrine way.     

Proposed mechanism for increased insulin-mediated glucose transport in adipose cells from young, obese Zucker rats. Large intracellular pool of glucose transporters

The mechanism for hyperresponsive insulin-mediated glucose transport in adipose cells from 30-day-old obese Zucker rats was examined. Glucose transport was assayed by measuring 3-O-methylglucose transport, and the concentration of glucose transporters was estimated by measuring specific D-glucose-inhibitable cytochalasin B binding. Insulin increased glucose transport activity by approximately 17 fmol/cell/min in cells from obese rats compared to 3 fmol/cell/min in lean littermates. Insulin increased the concentration of glucose transporters in the plasma membrane fraction by about 15 pmol/mg of membrane protein in both groups. The insulin-mediated decrease in the concentration of transporters in the low-density microsomal fraction was 30 pmol/mg of membrane protein for the obese rats compared to 15 pmol/mg of membrane protein for the lean controls. An estimated number of glucose transporters was calculated using membrane protein and enzyme recoveries for each group. Insulin increased the number of transporters in the plasma membrane by 3 X 10(6) sites/cell for the obese rats and only 0.6 X 10(6) sites/cell for the lean controls. In addition, insulin decreased the number of transporters/cell in the intracellular membrane pool by approximately 4 X 10(6) sites/cell for the obese rats and 0.9 X 10(6) sites/cells for the lean rats. The total number of transporters/cell was about 7 X 10(6) sites/cell for the obese animals and 1.6 X 10(6) sites/cell for the lean controls. In the basal state, more than 80% of these transporters were located in the intracellular pool for both the lean and obese rats. Thus, the marked hyperresponsive insulin-mediated glucose transport observed in adipose cells from 30-day-old obese Zucker rats may be the consequence of a marked increase in the number of glucose transporters in the intracellular pool.     

Glucose refractoriness of beta-cells from fed fa/fa rats is ameliorated by nonesterified fatty acids

The aim of this study was to characterize the glucose responsiveness of individual beta-cells from fa/fa rats under ad libitum feeding conditions. Enlarged intact islets from fed fa/fa rats had a compressed insulin response curve to glucose compared with smaller islets. Size-sorted islets from obese rats yielded beta-cells whose glucose responsiveness was assessed by reverse hemolytic plaque assay to determine whether glucose refractoriness was caused by a decreased number of responsive cells or output per cell. In addition, the effects of palmitic acid on glucose-stimulated insulin secretion were assessed because of evidence that nonesterified fatty acids have acute beneficial effects. Two- to threefold more beta-cells from >250 microm diameter (large) islets than <125 microm diameter (small) or lean islets responded to low glucose. Increasing the glucose (8.3-16.5 mM) induced a >10-fold increase in recruitment of active cells from small islets, compared with only a 2.6-fold increase in large islets. This refractoriness was partially reversed by preincubation of the cells in low glucose for 2 h. In addition, secretion per cell of the large islet beta-cell population was significantly reduced compared with lean beta-cells, so that the overall response capacity of large but not small islet beta-cells was significantly reduced at high glucose. Therefore, continued near-normal function of the beta-cells from small islets of fa/fa rats seems crucial for glucose responsiveness. Incubation of beta-cells from large islets with palmitic acid normalized the secretory capacity to glucose mainly by increasing recruitment and secondarily by increasing secretion per cell. In conclusion, these studies demonstrate refractoriness to glucose of beta-cells from large islets of fa/fa rats under ad libitum feeding conditions. When acutely exposed to nonesterified fatty acids, islets from fa/fa rats have a potentiated insulin response despite chronic elevation of plasma lipids in vivo.     

Changes of Islet Size and Islet Size Distribution Resulting from Protein-Malnutrition in Lean (Fa/Fa) and Obese (fa/fa) Zucker Rats

TSE, ELIZABETH O, FRANCINE M GREGOIRE, BRIGITTE REUSENS, CLAUDE REMACLE, JOSEPH J HOET, PATRICIA R JOHNSON, JUDITH S STERN. Changes of islet size and islet size distribution resulting from protein malnutrition in lean (Fa/Fa) and obese (fa/fa) Zucker rats. Potential alterations in islet size and islet size distribution resulting from protein malnutrition were studied in lean (Fa/Fa) and obese (fa/fa) Zucker rats. The purpose was to investigate whether the distribution of enlarged islets in obese rats was altered by low-protein feeding. Four-week-old, male, lean and obese Zucker rats were fed either a diet containing 20% (w/w) protein (control diet) or a diet containing 5% (w/w) protein (low-protein diet) for 3 weeks. Pancreata were dissected at autopsy and immunostained for insulin. Islet size and distribution were determined by morphometric analysis. Body-weight gain, food intake, and serum insulin and glucose were also measured. After 3 weeks on the diets, serum insulin was significantly lower in both lean (-75%) and obese (-54%) rats fed low protein compared with that in controls. However, obese rats were still hyperinsulinemic compared with lean rats. Protein malnutrition resulted in a shift in distribution of islets to smaller size both in lean and in obese rats, with an increase in the population of small islets (100 μm²) and a decrease in the population of large islets (>20,000 μ;m²). In lean and obese rats fed low protein, β-cell weight was significantly lower, B cell volume fraction tended to decrease, and islet number per section area was significantly elevated when compared with controls. Taken together, these results show that protein deficiency alters the endocrine pancreas in both lean and obese Zucker rats. Although the decrease in islet size and the shift in distribution to smaller islets most likely contribute to the decrease in serum insulin concentration, these changes appear insufficient to normalize hyperinsulinemia in the obese Zucker rat.     

Acute effects of exercise on circulating leptin in lean and genetically obese fa/fa rats

Mechanisms of regulation of plasma leptin in lean and genetically obese animals are not completely understood. In particular a relation has been proposed between energy metabolism and leptin. However, it is not clear how energy expenditure and leptin are related under exercise in lean and obese animals. To clarify these aspects we investigated lean and genetically obese (fa/fa) Zucker rats undergoing a single bout (30 min) of swimming and measured several biochemical and hormonal parameters of energy metabolism and leptin changes throughout the study. Moreover ob-gene expression in adipose tissue was also measured. Our results showed that plasma leptin is decreased by 30% at the end of exercise in lean animals while resulting unaffected in obese animals. Leptin changes in lean rats are concomitant with the peak of NEFA and glycerol release from adipose tissue rather than with the reduction of plasma insulin. Ob-gene expression in adipose tissue was markedly increased in fa/fa compared to lean rats, but was not modified by exercise both in lean and obese animals. In conclusion our data show that leptin changes during exercise are related to lipolytic events in adipose tissue and support a link between leptin and energy expenditure.     

Normal levels of serum corticosterone and hepatic glucocorticoid receptors in obese (fa/fa) rats

In an attempt to understand the hyper-responsiveness to glucocorticoids that is characteristic of genetically obese fa/fa rats, we have measured the levels of free corticosterone in serum from lean and obese rats as well as the number of cytosolic and "nuclear" binding sites in livers of these rats. Both the lean and obese rats had similar amounts of free corticosterone available for biological activity at 4 weeks and 10 weeks of age. Measurement of glucocorticoid binding to hepatic glucocorticoid receptors failed to show any differences between genotypes leading to the suggestion that the abnormal glucocorticoid response in obese rats may be due either to post-receptor defects or to a permissive action of the steroid in the expression of the fa/fa genotype.     

Reduced sensitivity to dexamethasone of pancreatic islets from obese (fa/fa) rats.

The direct effects of dexamethasone exposure on insulin secretion from islets of fa/fa rats and their lean littermates (Fa/?) were compared. After 72 h culture in 1 nM dexamethasone, glucose (27.5 mM)-stimulated insulin secretion over 90 min from islets of lean rats was significantly decreased compared with islets cultured without dexamethasone (12.9 +/- 1.4 vs. 5.7 +/- 1.0% of total islet content, p < 0.05). Higher doses of dexamethasone for 24-48 h culture produced similar effects. For islets of fa/fa rats, the minimum inhibitory concentration of dexamethasone was 10-fold higher, and islets required at least 48 h exposure for inhibitory effects to be observed. Dexamethasone also decreased the insulin response by islets to glybenclamide, indicating that dexamethasone effects were not specific to glucose transport or metabolism. The results suggest that islets of fa/fa rats may be less sensitive to direct inhibitory effects of glucocorticoids on glucose-stimulated insulin release than islets of lean animals.     

Evidence that non-covalent forces, thiol and disulphide groups affect the structure and binding properties of the prolactin receptor on hepatocytes from pregnant rats.

Incubation of hepatocytes from pregnant rats with dithiothreitol decreased specific 125I-prolactin (125I-prl) binding to such cells by about 20% relative to control. This was not due to a non-specific effect of dithiothreitol on the cell membrane, since reduction also altered the binding of prl to solubilized partially purified receptor. Exposure of hepatocytes to N-ethylmaleimide (6 mM) for periods as brief as 1 min decreased the subsequent specific binding of 125I-prl by more than 50%. N-Ethylmaleimide was less effective as an inhibitor of binding when applied after hepatocytes had been exposed to 125I-prl, binding being decreased by about 15%. Scatchard analysis demonstrated that the effect of N-ethylmaleimide resulted from loss of receptor-binding capacity without any substantial effect on the affinity of the prl receptor for hormone. Dithiothreitol diminished the affinity of lactogenic sites for prolactin without altering cellular binding capacity. These observations suggest that thiol and disulphide groups are present in the prl receptor and that these functional moieties regulate the formation and properties of prl receptor complexes. The species to which 125I-prl had bound were identified by affinity labelling. 125I-prl was covalently coupled into saturable complexes of Mr 65000 and 50000. 125I-human growth hormone (125I-hGH) was covalently incorporated into complexes of Mr 300 000, 220 000, 130 000, 65 000 and 50 000. Bovine growth hormone (bGH), but not prl, competed for 125I-hGH uptake into the 300 000-, 220 000- and 130 000-Mr complexes, indicating that these species were somatogenic. Prl, but not bGH, inhibited 125I-hGH uptake into 65 000- and 50 000-Mr complexes. This demonstrated that 125I-hGH in the presence of bGH could affinity-label lactogenic receptors. 125I-prl aggregates in Triton X-100, whereas 125I-hGH does not. Therefore lactogenic complexes to which 125I-hGH was bound in the presence of excess bGH were solubilized in Triton X-100 and characterized sequentially by gel filtration and affinity labelling. Prl receptors were eluted from columns of Sepharose 6B as a species of Mr380 000. Fractionation of the 380 000-Mr species on sodium dodecyl sulphate polyacrylamide gels resulted in the isolation of complexes of Mr 65 000 and 50 000. Thus non-covalent forces stabilize aggregates of the monomeric prolactin receptor.     

The effects of corticosterone, cold exposure and overfeeding with sucrose on brown adipose tissue of obese Zucker rats (fa/fa).

GDP binding to brown-adipose-tissue mitochondria was decreased in obese Zucker rats. Adrenalectomy restored both GDP binding and serum tri-iodothyronine of obese rats to values observed in lean rats. The effects of adrenalectomy on GDP binding and serum tri-iodothyronine were reversed by corticosterone. Decreasing food intake had no effect on brown-adipose-tissue GDP binding in obese rats. Young (5-week-old) obese rats showed a normal increase in brown-adipose-tissue mitochondrial GDP binding after housing at 4 degrees C for 7 days, but this response was attenuated in 10-week-old obese rats. Overfeeding with sucrose increased brown-adipose-tissue thermogenesis in lean, but not in obese, rats. After adrenalectomy, overfeeding with sucrose enhanced brown-adipose-tissue mitochondrial GDP binding in obese rats.