Marilyn Scott

In Vitro Insulin-Like Growth Factor-I, Growth Hormone, and Insulin Resistance Occurs in Symptomatic Human Immunodeficiency Virus-1–Infected Children

ABSTRACT: Poor growth is a common feature of symptomatic children (Centers for Disease Control stage P2) infected with human immunodeficiency virus-1 (HIV-1). However, several previous studies have failed to show any relationship between serum hormone levels and poor growth. To assess the roles of hormone deficiency and hormone resistance in the development of poor growth in HIV-1-infected children, we studied six asymptomatic Centers for Disease Control stage P1 [height SD score = 0.01 ± 1.0 (mean ± SD)], 10 P2 (height SD score = −2.0 ± 1.0), and six short, normal children (height SD score = −2.4 ± 1.2). Mean weight:height SD scores were similar in all three groups, suggesting that gross nutritional status did not differ between groups. There were no significant differences between groups with respect to mean plasma levels of IGF-I, thyroid hormones, TSH, and cortisol. As an index of hormone sensitivity, we quantified in vitro colony formation of erythroid progenitor cells, isolated from peripheral blood of study subjects, in response to IGF-I, growth hormone (GH), and insulin. P2 subjects had a quantitative mean reduction in erythroid progenitor cells colony formation in response to IGF-I of 32% compared with P1 subjects (p = 0.001 by analysis of variance) and 21% compared with controls (p = 0.006); in response to GH of 21% compared with controls (p = 0.015); and in response to insulin of 35% compared with P1 subjects (p = 0.038) and 34% compared with controls (p = 0.004). Similar qualitative differences (changes in shape) of the three hormone response curves between P2 and P1 and P2 and control subjects were observed. No differences in either quantitative or qualitative erythroid progenitor cells responses to IGF-I, GH, or insulin between P1 and control subjects were observed. We conclude that more severe HIV-1 infection in children is associated with in vitro resistance to the growth-promoting actions of IGF-I, GH, and insulin that is unrelated to the presence of gross malnutrition, differences in hematologic status, or over-whelming illness. This resistance to IGF-I could contribute to the poor in vivo growth seen in a symptomatic HIV-1-infected children.

Ontogeny of the mammalian insulin receptor: Studies of human and rat fetal liver plasma membranes

Abstract Insulin binding to human fetal plasma liver membranes was studied in preparations segregated into three pools according to length of gestation: 15–18 weeks (Pool A), 19–25 weeks (Pool B), and 26–31 weeks (Pool C). Receptor numbers, calculated by extrapolation of Scatchard plots to the X axis, increased from 25 × 1010 sites per 100 μg protein in the youngest group (Pool A) to 46 × 1010 sites per 100 μg protein in Pool B. No further increase in receptor number was seen in Pool C. The affinity constant for insulin at tracer concentrations, K e (“empty site”), was 1.53 × 108 M−1 in Pool A and was only slightly higher than K f (“filled site”). K e was higher in Pool B, 1.75 × 108 M−1, and in Pool C reached a value of 5.63 × 108 M−1. In Pool C K f was 2.3 × 108 M−1. Insulin binding of liver plasma membranes from rat fetuses aged 14, 16, 18, and 21 (term) days and adults was also studied. Maximum binding capacity tended to increase with gestational age and was 130 × 1010 sites per 100 μg protein at term, which was in excess of that found in adult rats (89–90 × 1010). In addition, K e increased from 0.75 × 108 M−1 at 14 days to 3.02 × 108 M−1 at term, a value higher than that found in pregnant and nonpregnant adults. Dissociation of insulin in the presence of high concentrations of insulin was significantly enhanced in tissues from 18-day and term fetuses and adults, but not in membranes from fetal rats aged 14 and 16 days. These data appear to indicate that site-site interactions are not present in early fetal existence. These changes in insulin binding with increased length of gestation are not ascribable to changes in relative proportions of hematopoietic and parenchymal tissue. Human fetal plasma liver membranes demonstrated elevated insulin binding with increased gestational age, but comparison of fetal and adult liver could not be done. However, newborn human infants have been shown to have a higher capacity for binding insulin to circulating monocytes than adults. Also, human fetuses apparently lack the capability to diminish monocyte receptors in the presence of hyperinsulinemia. These experiments show that an increase in insulin receptor binding capacity and affinity also occurs in the liver of the rat fetus at term as compared to the adult rat. The reasons and mechanisms underlying enhanced capacity for insulin binding by fetal and newborn members of human and rodent species are not known.

Stimulation of phosphatidylcholine synthesis by fatty acids in fetal rabbit type II pneumocytes

After 24 h exposure to 0.1 mM oleate or 0.1 mM palmitate there was a 2- and 1.7-fold increase, respectively, in the incorporation of choline into the lipids of type II pneumocytes. Palmitate increased the labeling of disaturated phosphatidylcholine (PC) from 23.0% of total labeled PC in control cultures to 56.6% and oleate decreased labeling of disaturated PC to 9.4%. The percentage of total cellular radioactivity found in the lipid fraction was also markedly higher in the fatty acid-treated cells (83.3% for oleate and 78.7% for palmitate) than in control cultures (64.0%). Radioactivity in water-soluble choline metabolites was correspondingly lower, with phosphocholine representing more than 95% of the label in both control and experimental cultures. After a 3 h pulse-chase period, oleate and palmitate significantly increased the percentage of total cellular radioactivity in PC and decreased the percentage in phosphocholine. Similar results were obtained by adding melittin (1-2 micrograms/ml) or phospholipase C (0.05 U/ml) to the culture medium. The stimulation of PC synthesis by fatty acids was demonstrated as early as 1 h after exposure to oleate or palmitate and at all concentrations from 0.025 to 0.25 mM. Cytidylyltransferase activity in total cell homogenates was also enhanced by long-term exposure to fatty acids and short-term addition of fatty acids or phospholipase C and melittin to the culture medium. A similar increase in cytidylyltransferase activity was found in the 100 000 X g particulate fraction of type II cells exposed to fatty acids, whereas no differences were found between the cytosolic fractions of control and treated cells. These results support the concept that an increase in intracellular level of fatty acids either from an exogenous source or following the activation of endogenous phospholipases regulates PC synthesis in fetal type II pneumocytes.

Effects of cyclic AMP analogues and phosphodiesterase inhibitors on phospholipid biosynthesis in fetal type II pneumocytes

Purified type II pneumocytes grown in monolayer cultures after isolation from fetal rabbit lung organotypic cultures were employed to investigate effects of cAMP analogues and phosphodiesterase inhibitors on [methyl-14C]choline and [9-10(n)3H]palmitate incorporation into cell lipids. After 24 h exposure to 0.5 mM N6,O2-dibutyryl-cAMP or 8-bromo-cAMP, a significant increase was found in the rate of incorporation of choline into phospholipids. Addition of 1 mM 1-methyl-3-isobutylxanthine or aminophylline also increased incorporation of choline into phospholipids but did not significantly change the incorporation of choline into sphingomyelin. These effects were not due to increased uptake of choline or changes in the pool size of the precursor. Cyclic AMP analogues also stimulated the rate of incorporation of palmitate into most lipid fractions but did not alter the relative percentages of incorporation of either precursor into any of the phospholipids. Phosphodiesterase inhibitors did not significantly change the rate of incorporation of palmitate into neutral lipids and most phospholipids, except for a decrease into sphingomyelin, phosphatidylinositol and phosphatidylethanolamine. However, they increased the percentage of incorporation of palmitate into phosphatidylcholine and decreased the percentage of incorporation into most other phospholipids. These data clearly indicate that cAMP can stimulate the synthesis of phospholipids within the type II pneumocytes. This effect is probably a general stimulation effect for the cAMP analogues but methylxanthines may selectively increase the synthesis of surfactant lipids such as phosphatidylcholine while decreasing that of other membrane-associated phospholipids.

Prevalence of three mutations in the Gsα gene among 24 families with pseudohypoparathyroidism type Ia

Pseudohypoparathyroidism type Ia (PHP-Ia), an inherited multi-hormone resistance syndrome, is associated with deficient cellular activity of the alpha-subunit of the guanine nucleotide-binding protein (Gs alpha) that stimulates adenylyl cyclase. We determined prevalence of three recently described mutations in exons 1 and 10 of the Gs alpha gene among 24 unrelated patients with PHP-Ia. Restriction analysis was used to detect two mutations that produce unique RFLPs, and allele-specific oligonucleotide hybridization was used to detect the other mutation. As none of these mutations were not found, genomic DNA was analyzed with denaturing gradient gel electrophoresis to screen for other mutations in exon 10. Mutations of the initiation codon and exon 10 in the Gs alpha gene thus rarely (< or = 4% each) cause PHP-Ia and the Gs alpha gene mutations causing PHP-Ia are heterogeneous and unique to each pedigree.

Use of In Vitro Clonogenic Assays to Differentiate Acquired From Genetic Causes of Insulin Resistance

Insulin resistance may be due directly to genetically programmed disorders of insulin action or acquired defects in which environmental factors influence insulin action. To address the issue of this distinction, we studied the ability of insulin to stimulate colony formation in primary cultures of erythroid progenitors (assumed to retain environmental influences) and immortalized T lymphocytes (presumed to reflect only genetic influences). Four patients with hyperinsulinemia and disturbed glucose metabolism were studied (2 patients with acanthosis nigricans, 1 of whom had circulating anti-insulin-receptor antibodies, 1 with partial lipodystrophy, and 1 with Cushings syndrome). The mean colony-forming ability of their erythroid progenitor cells in response to insulin stimulation (≤1.6 pM) was significantly blunted compared with control cells (P < 0.05). The mean responsiveness of their immortalized T-lymphoblast cell lines to similar insulin concentrations was no different than that of control T-lymphocyte lines, consistent with an acquired cause for the observed insulin resistance in each case. A T-lymphocyte line from a patient with leprechaunism, however, showed no stimulation in response to physiological concentrations of insulin. With these same in vitro methodologies, there was normal T-lymphocyte line responsiveness to insulinlike growth factor I (IGF-I) or insulin concentrations >8.6 pM; both of these responses could be completely blocked by preincubation with an antibody to the IGF-I receptor. These findings suggest that, despite resistance to physiological levels of insulin, the high circulating insulin concentrations present in the serum of these patients could mediate unwanted tissue-specific growth through an intact IGF-I receptor-effector mechanism.

Regulation of phospholipid synthesis by intracellular phospholipases in fetal rabbit type II pneumocytes.

Exposure of fetal type II pneumocytes to phospholipase A2 inhibitors led to significantly reduced choline uptake and decreased synthesis of total and disaturated phosphatidylcholines from both [methyl-14C]choline and [9,10(n)-3H]palmitate precursors. The percentage of the total synthesized phosphatidylcholine recovered as disaturated phosphatidylcholine was increased when compared to that in control cultures, suggesting that unsaturated phosphatidylcholine synthesis was reduced to a greater extent than that of the disaturated species. Synthesis of sphingomyelin and phosphatidylethanolamine from labeled palmitate was also reduced, whereas that of phosphatidylinositol and phosphatidylglycerol was significantly increased. Addition of phospholipase C resulted in increased synthesis of phosphatidylcholine from both labeled precursors; no significant changes were found in synthesis of most of the other 3H-labeled lipids. Added phospholipase A2 did not lead to any changes in either choline or palmitate incorporation. However, when melittin (a phospholipase A2 activator) was added to the cultures, greater incorporation of both palmitate and choline was observed, along with a significant increase in the percentage of total cellular radioactivity in 14C-labeled lipids, indicating also stimulation of phosphatidylcholine synthesis. A marked increase in CTP: phosphorylcholine cytidylyltransferase activity was found after treatment of the cultures with phospholipase C. Exposure to quinacrine also increased the activity of this enzyme. Addition of phospholipase C and melittin to prelabeled pneumocyte cultures accelerated degradation of cell phospholipids and the release of free fatty acids as the main degradation products. These findings suggest that intracellular phospholipases are regulators of synthesis of surfactant phospholipids in fetal type II pneumocytes, and that activation or inhibition of these phospholipases could represent a mechanism through which hormones and pharmacological agents modify surfactant and other phospholipid synthesis.

Triacetylated insulin: biologic activity and resistance to degradation.

Tritiated N-hydroxysuccinimide acetate was prepared with specific activities up to 5 Ci/mmole and utilized to prepare tritiated triacetyl insulin. Binding of triacetyl insulin to liver plasma membranes was measured by its capacity to displace 125I-monoiodoinsulin. At low concentrations, less than 10 ng/ml triacetyl insulin appears to be as effective as native insulin in reducing the binding of 125I-monoiodoinsulin to plasma membranes. At concentrations of 20 ng/ml and higher, triacetyl insulin is significantly less effective than native insulin in displacing binding of 125I-monoiodoinsulin to plasma membranes. The properties of triacetyl insulin in this system are not ascribable to deacetylation and conversion of the substituted product to native insulin. Biologic activity of triacetylated insulin was studied in two other in vitro systmes. A comparison was made of the capacity of native beef insulin and its triacetyl derivative to stimulate glucose oxidation by epididymal fat pads. At all three concentrations tested (2, 6, and 18 ng/ml), triacetyl insulin exerted considerable activity, although its potency was significantly less than that of native insulin. Similar effects were observed when biologic activity was measured by induction of tyrosine-alpha-ketoglutarate transaminase in a cultured liver cell system where significant activity of triacetyl insulin was found at concentrations of 10(-9)-10(-7) M. In all systems tested, the activity of triacetylated insulin could not be accounted for by deacetylation and conversion to native insulin. In all systems studied, triacetyl insulin was more resistant to degradation than was monoiodoinsulin.

IGF-I DOES NOT MEDIATE T-LYMPHOBLAST COLONY PROLIFERATION IN RESPONSE TO STEROID AND THYROID HORMONES: STUDIES IN CONTROL AND PYGMY T-CELL LINES.|[bull]| 518

The mechanism by which steroid hormones {estradiol [E2], testosterone [T], and 1,25(OH)2 Vitamin D3[1,25(OH)2D3]} and thyroid hormones [triiodothyronine(T3)] promote tissue growth is unknown, although, for E2 (acting on bone and uterus) and T3 (acting on bone), a role for local IGF-I has been suggested. We previously showed that HTLV-II-transformed T-cell lines from healthy adults augmented basal colony formation in response to peptide(GH, PTH, and ACTH) and glycoprotein (TSH) hormones through stimulation of local IGF-I. T-cell lines from African Efe Pygmies, however, were resistant to the direct and indirect growth-promoting actions of IGF-I. We, therefore, used these cell lines to determine the mechanism of T-cell growth in response to steroid and thyroid hormones. We quantified colony formation of American control T-cell lines (n=7) in the presence and absence of αIR-3 antibody against the type 1 IGF receptor and Pygmy T-cell lines (n=8) in response to E2 (36.7-1835 pmol/L), T (34.7-17,350 pmol/L), 1,25(OH)2D3 (2.4-24,000 pmol/L), and T3 (1536-192,000 pmol/L). There were no statistically significant differences by ANOVA in overall response curves for any of the four hormones comparing control clonal responses in the presence or absence of αIR-3, and no statistically significant difference in overall responses between control and Pygmy T-cell lines. From these data, we conclude that: (1) normal T-cell lines grow in response to E2, T, 1,25(OH)2D3, and T3; (2) these responses are not mediated through local IGF-I since they are unaffected by pretreatment with antibody against the type 1 IGF receptor; and (3) Pygmy T-cell lines, which are genetically resistant to IGF-I, grow equivalently to control T-cell lines in response to E2, T, 1,25(OH)2D3, and T3, further underscoring the IGF-I independence of this stimulation in our system. These findings, combined with our previous observations, indicate that T-cell growth in response to certain hormones that bind to cell-surface receptors is mediated by local IGF-I, whereas specific steroid and thyroid hormones that bind to cytoplasmic/nuclear receptors act by an IGF-I-independent mechanism.

874 INSULIN RECEPTORS IN CYSTIC FIBROSIS (CF): INCREASE IN RECEPTOR NUMBER MAY EXPLAIN INSULIN SENSITIVITY

Specific 125I insulin binding to circulating monocytes was studied in a group of 13 patients with CF, 4 of whom were insulin dependent. Nine untreated patients had mild carbohydrate intolerance in response to oral glucose as compared to controls (201±26mg/dl vs.103±6 M±SEm at 2h; 144±26 vs.92±5 at 3h). The peak insulin response was delayed to 2 hours in CF and lower than the one hour peak of normals (44mIU/ml vs.66mIU/ml). Calculated insulinogenic index, II (∑Δinsulin/∑Δglucose) was lower for CF than controls (0.21±0.05 vs.0.37±0.04 p<0.01), indicating enhanced insulin sensitivity. Scatchard analysis of insulin binding revealed a marked increase in receptor sites in untreated CF patients as compared to controls; 44,000 sites/cell vs. 25,000 sites/cell. Specific insulin binding at tracer concentrations (0.3ng/ml) was lower in CF 3.14% vs.5.14%, and the calculated affinity constant for binding (Ke) was reduced, 0.8x108 M−1 vs. 2.5x108 M−1. Insulin treated patients did not have lower receptor numbers or altered affinity as compared to the untreated. Thus 1) in CF diminished insulin secretion is associated with an increased number of receptor sites 2) increased binding sites would explain sensitivity to endogenous insulin, as demonstrated by the decreased II as well as to exogenous insulin (reported by others) 3) the consequences of the substantial increase in receptor number may be offset somewhat by reduced receptor affinity.