Studies of insulin binding in children using human erythrocytes in small amounts of blood.

We have demonstrated specific insulin binding by the erythrocytes (RBCs) of children. Complete binding studies were done using as little as 5 ml of blood. The receptors exhibited competition-inhibition curves and nonlinear Scatchard plots similar to those reported for insulin target tissues, such as the hepatocyte and the adipocyte. Compared with those from adults, the RBCs from children had significantly greater numbers of insulin receptors per cell (P less than 0.05). The total insulin bound by the RBCs from both children and adults, however, was not different over the physiologic range of insulin concentrations. Cord blood RBCs showed greater numbers of receptors per cell than did those from either children or adults; however, the affinity for insulin was similar in both groups. The total amount of insulin bound by cord blood was significantly greater than that in either children (P less than 0.01) or adults (P less than 0.05) over the physiologic range of insulin concentrations. The method used to measure insulin binding by erythrocytes and relatively little intra- and interassay variability, and there was little diurnal variation in binding. Storage of heparinized blood at 4 degree C for 24--36 h had no effect on insulin binding by the RBCs. We conclude that the measurement of insulin binding by RBCs from small volumes of blood may be particularly useful in the study of infants and children with disorders of carbohydrate metabolism to elucidate the role, if any, of abnormal receptor function in their condition.

Increased integrated concentration of norepinephrine, epinephrine, aldosterone, and growth hormone in patients with uncontrolled juvenile diabetes mellitus.

The 24-h integrated plasma concentration of glucose (IC-glucose), norepinephrine (IC-NE), epinephrine (IC-E), cortisol (IC-F), growth hormone (IC-GH), aldosterone (IC-ALDO), and plasma renin activity (IC-PRA) were measured in 11 nonobese juvenile-onset nonketotic diabetic patients exhibiting hyperglycemia and glycosuria and 34 matched control subjects using a portable pump, drawing blood at a constant rate through a nonthrombogenic i.v. catheter. The diabetic patients had a noticeable rise of their IC-NE, IC-E, IC-GH, and IC-ALDO. There was no significant difference between the IC-F and IC-PRA of the patients and the control subjects.

Ontogeny of erythrocyte insulin binding in the sheep.

The ontogeny of insulin binding in the sheep was studied using the erythrocytes (RBCs) of 31 fetuses, 10 lambs, and 5 adult animals. Six fetuses were studied on three occasions over a 2-week period from 120--135 days of gestation to provide longitudinal data on changes in insulin binding. Maximal percent binding of [125I]iodoinsulin and receptor concentration decreased significantly as the age of the animal increased (r = 0.76, P less than 0.001 and r = --0.49, P less than 0.001, respectively). Total loss of insulin binding to RBCs was estimated to occur in the second postnatal month, and the RBCs from the adult sheep showed no specific insulin binding. The osmotic fragility of RBCs in each developmental group of animals was also studied to assess possible differences in RBC membrane properties. RBC osmotic fragility was significantly lower in fetuses than in adult sheep (osmotic fragility 50 = 0.55% phosphate-buffered saline vs. 0.76% phosphate-buffered saline, respectively; P less than 0.001). The data suggest that fetal RBCs of lower osmotic fragility and high insulin binding capacity are progressively replaced during late prenatal and early postnatal life by adult-type RBCs of increased osmotic fragility and lacking binding capacity for insulin. The timing of the disappearance of insulin binding to RBCs coincides with the final transition in the animals from a monogastric to a ruminant metabolic state, and may reflect a change in the need for insulin with age.

Erythrocyte insulin binding in obese children and adolescents.

Insulin binding studies in obese patients have been limited to adults due to the relative inaccessibility to tissues for study in the pediatric age group. Insulin binding to the erythrocytes (RBCs) of 9 obese pre-pubertal children, 8 obese adolescents, and 10 obese adults was studied. There was a mean decrease of 15% of insulin binding in the obese patients (P < 0.02 vs. controls). Calculation of receptor concentrations by means of Scatchard plots showed a mean 30% reduction in insulin receptors on the RBCs of obese patients as a group (P < 0.001 vs. controls). The binding of insulin and receptor concentration were inversely proportional to the fasting plasma insulin concentration (= -0.60 and -0.44, respectively). These correlations were significant (P < 0.001 and P < 0.05, respectively). The mean empty site receptor affinity (Ke) was significantly increased in obese patients, but only partially compensated for the loss of receptors with respect to total insulin bound over the physiological range of insulin concentrations. The results of binding studies in the obese adults were similar to those in the children and adolescents, and agreed with published reports of insulin binding in obese adults using adipocytes or monocytes as the source of insulin receptors. The observed decrease in insulin binding to the RBCs of obese children and adolescents correlated with fasting hyperinsulinemia and, therefore, may contribute to the etiology of the insulin resistance or glucose intolerance observed in these patients.

Suppression of gonadotropin secretion by a long-acting gonadotropin-releasing hormone analog (leuprolide acetate, Lupron Depot) in children with precocious puberty.

The GnRH analog leuprolide acetate has been shown to be effective in the treatment of precocious puberty when given as a daily sc injection. We studied the effectiveness of a single im dose of a new depot form of leuprolide in suppressing estradiol and gonadotropin secretion in children with precocity. Five girls with previously untreated precocity showed significant decreases in basal serum estradiol and FSH levels and in peak LH levels (after GnRH testing) 30 days after a single im dose of leuprolide acetate for depot suspension. Mean peak FSH levels also fell greatly, but the difference was not significant. No adverse effects were noted during the first 4-6 months of monthly im injections. Depot im leuprolide appears to be effective in suppressing estradiol and gonadotropin secretion, and may be a useful method of treating children with central precocious puberty.

Insulin binding to monocytes in obese patients treated with carbohydrate restriction and changes in physical activity.

Mean [125I]insulin binding to circulating monocytes was low (P less than 0.05 compared to normal controls) in nine obese patients on a weight-maintaining diet in which 45% of the calories were carbohydrate. On a 10% carbohydrate diet, insulin binding was normal in six of seven obese patients. Plasma insulin concentrations were elevated in obese patients on both diets. No correlation was found between insulin binding and plasma insulin concentration. Glucose intolerance and hyperinsulinemia were worsened by keeping the patients at rest and were improved by having the patients walk 3-4 miles/day. The change in physical activity had no effect on [125I]insulin binding to monocytes. We conclude that 1) insulin binding to monocytes in obese patients is generally low in patients on a carbohydrate-rich diet, but is normal in patients on carbohydrate-restricted diets; 2) down-regulation of insulin receptors does not necessarily occur in the presence of hyperinsulinemia: and 3) a walking program results in an improvement in glucose tolerance and hyperinsulinemia that is not associated with a change in insulin binding.

Studies of 3 beta-hydroxysteroid dehydrogenase genes in infants and children manifesting premature pubarche and increased adrenocorticotropin-stimulated delta 5-steroid levels.

Classic 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) deficiency congenital adrenal hyperplasia (CAH) results from a mutation in the type II 3 beta HSD gene encoding adrenal and gonadal 3 beta HSD. We investigated the type II and type I 3 beta HSD gene sequences in 15 infants and children with premature pubarche (PP; mean/range of age at PP, 4/0.08-9 yr) and elevated ACTH-stimulated delta 5 precursor steroid levels. Compared to Tanner I control subjects of similar age, ACTH-stimulated hormonal levels were at 2.3-10.7 SD for 17-hydroxypregnenolone (delta 5-17P) in all PP subjects, at 2.2-17 SD for dehydroepi-androsterone (DHEA) and 2.4-5.6 SD for the delta 5-17P/cortisol (F) ratio in all PP subjects except 1 infant, and at 2.3-10 SD for the DHEA/ androstenedione (delta 5-A) ratio in 8 PP subjects. Compared to Tanner II normal children, the hormonal levels were at 3-8 SD for delta 5-17P in all 13 PP children, at 2.3-4.7 SD for the delta 5-17P/F ratio in 6 PP children, and at 2.3-6.5 SD for DHEA and 3.5-9 SD for the DHEA/delta 4-A ratio in 7 PP children. Type II 3 beta HSD gene sequences, including regions of a putative promoter, all exons (I, II, III, and IV), and exon-intron boundaries, were normal in all subjects. Sequences of the type I 3 beta HSD gene encoding extraadrenal and extragonadal 3 beta HSD were normal in the 6 patients tested. The ACTH-stimulated delta 5-17P levels and delta 5-17P/F ratios in the PP children without type II 3 beta HSD gene mutation were exceedingly lower than the respective reported hormonal data for children with 3 beta HSD deficiency CAH with proven type II 3 beta HSD gene mutation. The ACTH-stimulated DHEA levels and DHEA/delta 4-A ratios were not exceedingly different between the children with and without type II 3 beta HSD gene mutation. These findings suggest that the degree of ACTH-stimulated delta 5 precursor steroid abnormality, such as delta 5-17P levels up to 10 SD above the normal mean level found in our PP patients, is not caused by a mild variant of 3 beta HSD deficiency CAH resulting from type II or type I 3 beta HSD gene mutation. The hormonal criterion for ACTH-stimulated delta 5-17P levels in patients with mild variant 3 beta HSD deficiency, therefore, is predicted to be higher than 10 SD above the normal mean value.

Low-dose intravenous insulin infusion in patients with diabetic ketoacidosis: biochemical effects in children.

Twenty-five episodes of diabetic ketoacidosis in 20 children were treated with continuous low-dose intravenous insulin infusion. Stable serum immunoreactive insulin concentrations were produced, along with prompt falls in glucose, beta-hydroxybutyrate, and glucagon levels, and a steadily increasing bicarbonate level. Neither hypokalemia nor hypophosphatemia developed. Elevated serum alanine concentrations were found during ketoacidosis in contrast to the lowered concentrations found in adults, and were correlated inversely with plasma glucagon concentrations. The treatment regimen described is safe, easy to use, efficacious, and resulted in prompt correction of the observed biochemical alterations in children with diabetic ketoacidosis.

Erythrocyte insulin binding in insulin-dependent diabetes mellitus: lack of relationship to duration and control of diabetes in children and adolescents.

Insulin binding was measured in the erythrocytes (RBCs) of four children and 12 adolescents with insulin-dependent diabetes mellitus in the basal (fasting, nonketotic) state. Children and adolescents with insulin-dependent diabetes mellitus showed normal binding of insulin to their RBCs when expressed as the total insulin bound over the physiologic range of insulin concentrations. The insulin receptor concentration and receptor binding affinity for insulin were also normal. These parameters of insulin binding were not correlated with either the duration of diabetes or the degree of diabetic control in the patients. Since insulin binding by erythrocytes has been shown to reflect binding by traditional target tissues (liver, fat), the data suggest that alterations in binding of insulin to cells in children and adolescents with insulin-dependent diabetes mellitus probably play little, if any, role in the response of these patients to exogenous insulin or in the control of their glucose metabolism in the basal state.

Adult-level insulin binding is present in term fetal rat CNS membranes.

Insulin binding was measured using partially-purified membranes prepared from the brains of term fetal and adult rats as the source of receptors. The membranes from the fetal rat brains bound at least as much insulin as did those from the adult rats at all concentrations of insulin used. This finding supports our hypothesis that insulin contributes to the postnatal growth of the rat brain.

Insulin inhibits pyramidal neurons in hippocampal slices.

Recent studies have confirmed the presence of insulin receptors in the rat brain although their function has still not been well defined. The present study explores the possibility that insulin receptors in the brain can alter or contribute to central neurotransmission. Insulin caused a dose-dependent inhibition of hippocampal pyramidal neurons. The pattern of inhibition mirrored the binding kinetics of insulin in the hippocampus. Two related peptides, proinsulin and desoctapeptide insulin, had neuronal effects consistent with their binding to insulin receptors in the brain. Proinsulin was effective in doses 30-fold greater than insulin, whereas desoctapeptide insulin had little or no effect. These observations indicate that the inhibitory effect of insulin in this tissue may be insulin receptor-mediated and support a previously suggested functional role of insulin in the central nervous system.

Developing rat brain binds monoiodinated insulin isomers similarly to other extrahepatic target tissues.

We recently reported a series of binding and metabolic studies which led to the conclusion that the developing rat brain is a target tissue for insulin. Since insulin target tissues (extrahepatic) are capable of differentiating between various monoiodoinsulin isomers, we measured the binding of the B26 monoiodoinsulin isomer compared to the A14 in newborn rat brain preparations to determine if the developing rat brain shared the same relative binding of these isomers (viz. B26 greater than A14) with other extrahepatic tissues. The B26 isomer bound 1.57, 1.50 and 1.34 times as much as did the A14 to brain membranes, glia and neurons, respectively, whereas both isomers were bound equally by liver plasma membranes. Competition-inhibition curves were generated using homologous unlabeled (127I) insulin isomers. Binding of the B26 isomer was greater than the A14 at all concentrations. Scatchard plots showed that the receptor concentrations for the two isomers were similar, and affinity profiles showed that the differences in binding could be accounted for by the greater affinity of the receptors for the B26 isomer. The results indicate that the developing rat brain shares with other extrahepatic insulin target tissues a greater affinity for B26 monoiodoinsulin isomer compared to A14. Future studies of insulin binding should avoid using mixtures of iodinated insulins so that a uniform interpretation of data is made possible.

Developing therapeutic relationships in multicultural settings.

The expertise of native peoples as well as resources drawn from humanities, education, and arts organizations in Arizona contributed to an educational program to heighten the cultural sensitivity of health care professionals at the Children's Health Center of St. Joseph's Hospital. The sentiment expressed by Kraut that "respect for the cultural preferences and taboos of more traditional medical systems thus renders American physicians more effective and in the long run more efficient" is supported by our experience with this program.

Growth hormone deficiency associated with methylmalonic acidemia.

Poor growth is a common finding in patients with organic acidemias. Growth hormone (GH) therapy has been considered in the management of these disorders as a mode to enhance anabolism and lower the high levels of methylmalonic acid. We report two patients with methylmalonic acidemia (mut(o)) and GH deficiency. Both patients had persistently elevated serum concentrations of methylmalonic acid, which failed to respond to conventional therapy. In anticipation of using GH therapy to reduce high methylmalonic acid concentrations, the first patient underwent GH testing utilizing a provocative glucagon stimulation test and was found to be deficient. He was subsequently treated with GH and demonstrated improved growth, but his methylmalonic acid concentrations remain elevated. The second patient was also found to be GH deficient. These findings suggest that GH deficiency may be an etiologic factor in the poor growth seen in patients with organic acidemia.

Insulin binding is a specific marker of fetal erythrocytes in ruminants.

The ability of erythrocytes (RBC) from sheep and cattle of various gestational and postnatal ages to bind insulin specifically was studied. Insulin binding to RBC decreased as gestational and postnatal age advanced and was absent in blood obtained from adult animals. Maximal percentage 125I-insulin bound to RBC (3.6 X 10(9)/ml) was highest in the fetuses of sheep and cattle (7.3 +/- .6 and 7.8 +/- .9, respectively) compared to postnatal animals (2.3 +/- .2 and 2.2 +/- .3, respectively), or adults (no binding) of the same species. The decrease in binding began antenatally, and binding was projected to be insignificant by the end of the second postnatal month. Most of the observed decrease was due to a progressive decrease in the number of receptors on the cell surface. The time course of this phenomenon, as well as the total absence of insulin receptors on the RBC of adult ruminants, provides independent evidence that two distinct populations of RBC in ruminants exist. The gradual appearance of the adult RBC with no insulin binding results in a decrease in observed binding to RBC in a given blood specimen as fetuses and postnatal animals age.

Advances in the treatment of precocious puberty.

The development of immunoassay techniques for accurately measuring serum gonadotropins and gonadal steroids allows distinction between central (GnRH-dependent) and peripheral (GnRH-independent) precocious puberty in children. Previously (see case 3) it was not possible to do this, and a rational approach to therapy could not be designed. Significant advances in the synthesis of superagonists of gonadotropin-releasing hormone have allowed effective treatment for children with CPP. Such treatment has only been possible for the past 10 to 15 years, and GnRHa have been approved for use in the United States for this indication only since 1992. Long-term studies are still needed to determine what effect, if any, such treatment has on fertility, because only a few patients, even from the earliest studies, have reached child-bearing age. All indications, however, suggest that treatment with GnRHa is reversible, as well as safe and efficacious. More challenging is the effective treatment of PPP, for which innovative approaches involving inhibition of gonadal steroid synthesis or action have met with some success. Again, long-term studies or efficacy and safety are needed.

Advances in the recognition and treatment of endocrine complications in children with chronic illness.

Children with chronic illness live with the specific consequences of their illness, as well as secondary endocrine abnormalities that further compromise growth and pubertal development. These secondary abnormalities may significantly add to their physiologic and psychological burden. Although these endocrine abnormalities theoretically arise as adaptations to the chronic illness, they may have deleterious effects if they persist untreated. Children with HIV infection and other wasting disorders, for example, show growth suppression out of proportion to the severity of their primary illness as a result of growth hormone resistance and enhanced cortisol secretion. In hematologic conditions such as sickle cell anemia, thalassemia, or bone marrow transplant, damage to the hypothalamus and/or pituitary may lead to growth hormone deficiency, gonadal insufficiency, and hypothyroidism. Growth and pubertal delay are also common among children with cystic fibrosis, along with insulin-dependent diabetes mellitus caused by pancreatic fibrosis. Similarly, children receiving long-term steroid therapy have delays in growth and pubertal development, accompanied by risk for osteoporosis, whereas chronic renal disease is associated with growth and pubertal delay, as well as secondary hyperparathyroidism. Recognition of potential endocrinopathies in children with chronic illness is an important aspect of the care of these children because the disturbances are frequently amenable to treatment, permitting full or partial restoration of normal growth and development in these children. In this chapter, the endocrine consequences of common chronic conditions of childhood are reviewed, as well as the etiology of the endocrine disturbance, the clinical consequences, and recommendations for treatment.

Low-dose intravenous insulin: treatment of diabetic ketoacidosis in children (suitability for use in community hospitals).

A regimen is described to simplify current methods of ketoacidosis therapy by using only standard intravenous equipment and without requiring albumin administration. After an initial intravenous insulin bolus of 2 units, a dose of up to 3 units per hour was sufficient to bring patients out of ketoacidosis. No hypoglycemia, significant hypokalemia, or rapid fall in serum osmolality was detected. Patients showed steady increases in endogenous bicarbonate and were out of metabolic acidosis in 8.4 hours. This modified regimen appears to be a safe and easy method of treating diabetic ketoacidosis in children and is suited for use in community hospitals. Since the method is described for children over the age of three or those not in severe coma, consultation should be sought for any infant and for any child in severe coma or with a significant complication, such as severe infection.

Diabetes in pregnancy: rationale and guidelines for care.

The management of pregnancy in a woman with diabetes is directed toward insuring a normal outcome for the mother and her newborn baby. There is ample evidence from studies in both humans and animals that maintenance of a normal maternal metabolic environment, particularly blood glucose concentrations, can avoid or ameliorate the complications of pregnancy responsible for increased morbidity and mortality in the mother and her baby. It is essential that this metabolic normalcy be achieved preconceptionally if congenital malformations, the leading cause of death in IDMs, are to be avoided. Thus, preconceptional care, intensive regulation of maternal glucose metabolism and fetal surveillance throughout pregnancy are critical. The use of the multidisciplinary team to optimize care has led to significant reductions in maternal and perinatal morbidity/mortality in IDMs, and is thus essential to successful outcomes in pregnancies complicated by diabetes.

Insulin is a growth-promoter of the developing brain: possible implications for the infant of the diabetic mother.

The combination of diabetes mellitus and pregnancy was once associated with significantly increased morbidity and mortality for mother and baby. Advances in the obstetric management of the mother with diabetes and parallel advances in the management of the infant of the diabetic mother (IDM) have lowered the morbidity and mortality in many centers to near those for pregnancies in the non-diabetic woman. A significant increase still exists in the prevalence of congenital malformations in IDMs. Recent animal studies have shown that these malformations are related to maternal hyperglycemia early in the pregnancy, and that if it is controlled with insulin during the critical period of embryologic development, then the malformations may be prevented.