High maternal iron status, dietary iron intake and iron supplement use in pregnancy and risk of gestational diabetes mellitus: a prospective study and systematic review.

AIM: High iron measured using dietary intake and biomarkers is associated with Type 2 diabetes. It is uncertain whether a similar association exists for gestational diabetes mellitus. The aim of this systematic review was to conduct a cohort study examining first trimester body iron stores and subsequent risk of gestational diabetes, and to include these findings in a systematic review of all studies examining the association between maternal iron status, iron intake (dietary and supplemental) and the risk of gestational diabetes. METHODS: Serum samples from women with first trimester screening were linked to birth and hospital records for data on maternal characteristics and gestational diabetes diagnosis. Blood was analysed for ferritin, soluble transferrin receptor and C-reactive protein. Associations between iron biomarkers and gestational diabetes were assessed using multivariate logistic regression. A systematic review and meta-analysis, registered with PROSPERO (CRD42014013663) included studies of all designs published in English from January 1995 to July 2015 that examined the association between iron and gestational diabetes and included an appropriate comparison group. RESULTS: Of 3776 women, 3.4% subsequently developed gestational diabetes. Adjusted analyses found increased odds of gestational diabetes for ferritin (OR 1.41; 95% CI 1.11, 1.78), but not for soluble transferrin receptor (OR 1.00; 95% CI 0.97, 1.03) per unit increase of the biomarker. Two trials of iron supplementation found no association with gestational diabetes. Increased risk of gestational diabetes was associated with higher levels of ferritin and serum iron and dietary haem iron intakes. CONCLUSIONS: Increased risk of gestational diabetes among women with high serum ferritin and iron levels and dietary haem iron intakes warrants further investigation.

Gestational diabetes: a red flag for future Type 2 diabetes in pregnancy? A retrospective analysis.

AIMS: This study sought to understand the relationship between Type 2 diabetes in pregnancy and previous gestational diabetes (GDM), and determine whether a previous pregnancy with GDM was associated with subsequent better pregnancy planning. METHODS: A retrospective review of medical records of women with Type 2 diabetes in pregnancy was conducted at three teaching hospitals to ascertain whether they had earlier GDM, and to determine whether this is associated with differences in measures of pregnancy planning and diabetes management. RESULTS: Of 172 index pregnancies affected by Type 2 diabetes, in 76 (44%) cases, the mother had a previous history of GDM. Within this cohort, a diagnosis of 'overt diabetes in pregnancy', made on the basis of a GTT result during pregnancy in the WHO diabetic range with persistent diabetes post partum, was more common among women who had previous GDM than women who had not had GDM (20% vs 7%, P = 0.02). Women who previously had GDM did not exhibit a higher incidence of preconception planning or folate supplementation. CONCLUSIONS: It is common for women with Type 2 diabetes in pregnancy to have had GDM previously. The diagnosis of GDM is an opportunity to improve future pregnancy planning and outcomes for women with Type 2 diabetes in pregnancy. This goal is yet to be realized.

Occurrence and recurrence of diabetes in pregnancy.

AIMS: To determine occurrence and recurrence rates of gestational diabetes among women having at least two consecutive pregnancies. Risk factors for recurrence of gestational diabetes and rates of second/third pregnancy pre-existing diabetes mellitus were also assessed. METHODS: Population-based study using longitudinally linked hospital discharge and birth records (2001-2009) in NSW, Australia. Participants included women without a pre-existing diagnosis of Type 1 or Type 2 diabetes at time of first pregnancy and with at least a first and second birth. Factors associated with recurrence of gestational diabetes were examined using multivariate log-binomial models to adjust for correlation within mothers and estimate relative risks and 95% confidence intervals. RESULTS: First occurrence of gestational diabetes was 3.7% (5315/142 843) in the first pregnancy and 2.7% (3689/137 528) in the second pregnancy. The recurrence rate of gestational diabetes in a second consecutive pregnancy was 41.2%. Risk of pre-existing diabetes in a pregnancy subsequent to one with first occurrence of gestational diabetes was 2.2% and 2.0% in the second or third pregnancy, respectively. Among women with a diagnosis of gestational diabetes in the first pregnancy, independent predictors of gestational diabetes recurrence were maternal age ≥ 35 years, ethnicity (Middle East/North Africa and Asia), pregnancy hypertension, large for gestational age infant and preterm birth in the first pregnancy, longer inter-pregnancy birth interval and pregnancy hypertension and multiple pregnancy in the second pregnancy. CONCLUSIONS: Gestational diabetes in a previous pregnancy is a strong indicator of future risk and a useful clinical marker for identifying women at elevated risk in a subsequent pregnancy.

Perinatal risk factors for early onset of Type 1 diabetes in a 2000-2005 birth cohort.

AIMS: To examine perinatal risk factors for the onset of Type 1 diabetes before 6 years of age, in a 2000-2005 Australian birth cohort. METHODS: Data from longitudinally linked delivery and hospital admission records (until June 2007) were analysed. Diabetes in mothers and children was identified from International Classification of Diseases 10 diagnosis codes in the hospital records. RESULTS: There were 272 children admitted to hospital with a first diagnosis of diabetes out of 502 040 live births. Incidence for the infants born in 2000 was 16.0 per 100 000 person-years. Maternal Type 1 diabetes was a significant risk factor [crude relative risk (RR) 6.33], but maternal Type 2 diabetes and gestational diabetes were not significantly associated with diabetes in the child. Late preterm birth (34-36 weeks) (RR 1.64) and caesarean section (RR 1.30) increased the risk of a diabetes admission. Size-for-gestational-age was significantly associated with onset of diabetes (small-for-gestational age RR 0.48), but neither birth weight categories nor birth weight as a continuous variable were associated with risk of diabetes. Increasing maternal age was associated with an increased risk of diabetes in the child (RR 1.13 for each additional 5 years of age). CONCLUSIONS: This study identified risk factors associated with onset of Type 1 diabetes before 6 years of age, in a recent birth cohort. Size-for-gestational-age had a consistent association with risk of early onset of Type 1 diabetes, small size being protective. Size-for-gestational-age measures should be preferred to birth weight thresholds when assessing risk of diabetes.

Maternal vitamin D deficiency, ethnicity and gestational diabetes.

AIMS: Vitamin D deficiency has been linked to impaired glucose metabolism. We determined whether serum 25-hydroxyvitamin D (25OHD) is associated with glucose metabolism in pregnant women and the effect of ethnicity on this relationship. METHODS: We analysed serum 25OHD concentrations in 307 pregnant women attending a metropolitan obstetric clinic between October 2003 and May 2005. Measurements from 264 of the women were taken at the time of glucose tolerance testing at mid-gestation, a population therefore at increased risk for gestational diabetes. Pearson correlation analysis was used to test for univariate linear relationships between the natural log of serum 25OHD (ln-25OHD) and other variables. Multiple regression analysis was used to adjust for confounding factors. RESULTS: Mean serum 25OHD concentration was 53.8 +/- 23.9 nmol/l (sd). Ln-25OHD was negatively correlated with serum parathyroid hormone as expected (r -0.24, confidence intervals -0.35 to -0.12). Ln-25OHD was also negatively correlated with fasting plasma glucose (r-0.20, -0.31 to -0.08), fasting insulin (r -0.20, -0.31 to -0.08) and insulin resistance as calculated by homeostasis model assessment (r -0.21, -0.32 to -0.09). The association between fasting glucose and log-transformed 25OHD concentration was of borderline significance after accounting for ethnicity, age and body mass index in multivariate analyses (-0.13, -0.26 to 0.01). The odds ratio of gestational diabetes in women with 25OHD < 50 nmol/l did not reach statistical significance (1.92, 95% confidence interval 0.89-4.17). CONCLUSIONS: Maternal 25OHD concentrations are inversely related to fasting glucose, although further studies are required to establish whether this is independent of the effects of ethnic background.

Outcomes of pregnancies in women with pre-gestational diabetes mellitus and gestational diabetes mellitus; a population-based study in New South Wales, Australia, 1998-2002.

AIM: To determine population-based rates and outcomes of pre-gestational diabetes mellitus (pre-GDM) and gestational diabetes mellitus (GDM) in pregnancy. METHODS: This was a cross-sectional study, using linked population databases, of all women, and their infants, discharged from hospital following birth in New South Wales (NSW) between 1 July 1998 and 31 December 2002. Women with, and infants exposed to pre-GDM or GDM were compared with those without diabetes mellitus for pregnancy characteristics and outcomes. RESULTS: Women with a singleton pregnancy (n = 370,703) and their infants were included: 1248 women (0.3%) had pre-GDM and 17,128 (4.5%) had GDM. Of those women with pre-GDM, 57% had Type 1 diabetes, 20% had Type 2 diabetes and for 23% the type of diabetes was unknown. Major maternal morbidity or mortality was more common in women with pre-GDM (7.9%) [odds ratio (OR) 3.2, 95% confidence interval (CI) 2.6, 3.9] and in women with GDM (3.1%) (OR 1.2, 95% CI 1.1, 1.4) when compared with women without diabetes (2.6%). Major infant morbidity or mortality occurred more frequently in infants exposed to pre-GDM compared with no diabetes (13.6% vs. 3.1%) (OR 5.0, 95% CI 4.2, 5.8) and in infants exposed to GDM compared with no diabetes (3.2% vs. 2.3%) (OR 1.4, 95% CI 1.3, 1.5). CONCLUSIONS: Pre-GDM and GDM continue to be associated with an increased risk of adverse maternal and neonatal outcomes; however, women with GDM have adverse outcomes less frequently. Rates of GDM and pre-GDM appear to be increasing over time. Clinicians should consider the potential for adverse outcomes, and arrange referral to appropriate services.

Induction of the insulin proreceptor by hydrocortisone in cultured lymphocytes (IM-9 line).

Hydrocortisone increases the number of insulin receptors at the surface of human cultured lymphocytes (IM-9 line) without altering the degradation of the mature receptor subunits. To elucidate the effect of glucocorticoids on the biosynthesis of the insulin receptor of IM-9 cells, we preincubated cells in the presence or absence of hydrocortisone (1.4 X 10(-6) M) and measured the incorporation of radiolabeled sugars into the insulin receptor components. From 6 to 22 h, there was a progressive increase in the incorporation of [3H]mannose into the insulin proreceptor (190,000 mol wt) and the mature subunits (210,000, 135,000, and 95,000 mol wt). The amount incorporated into hydrocortisone-treated cells was always three to four times higher than in control cells, despite no change in cell number, viability, or radioactive sugar pool. To test directly the earliest effect of hydrocortisone, we undertook pulse-chase studies. The incorporation of [3H]mannose into the insulin receptor precursor and the mature subunits was detectable as early as 30 min of chase and was two to three times higher in hydrocortisone-treated cells at any time point of incubation. In both groups, the increase into the proreceptor (190,000 mol wt) peaked by 60 min and decreased rapidly thereafter (half disappearance rate, 45 min); there was a sustained increase of incorporation into the two major mature subunits (135,000 and 95,000 mol wt) throughout the 4-h chase. Hydrocortisone represents the first pharmacologic agent shown to induce the synthesis of the insulin proreceptor. Further, we present a model system designed to study other agents that may act at a very early step in insulin receptor biosynthesis.

Insulin receptor of human cerebral gliomas. Structure and function.

The insulin receptor from human brain tumors of glial origin was examined for the first time using intact cells (from an established cultured human glioblastoma cell line) and partially purified solubilized membranes (from cultured cells and freshly isolated human brain tumors). The structure of the glial insulin receptor subunits was assessed by affinity cross-linking of 125I-insulin with the alpha-subunit of the receptor, neuraminidase treatment of the cross-linked receptor, behavior of the receptor on lectin columns, and electrophoretic mobility of the phosphorylated beta-subunit. The functions of the insulin receptor were examined by measuring specific 125I-insulin binding (receptor concentration, affinity, specificity, pH-, time-, and temperature dependence), insulin-induced down-regulation of the receptor, insulin-stimulated autophosphorylation of the beta-subunit, and phosphorylation of exogenous substrates as well as insulin-stimulated glucose uptake in glioblastoma cells. All of these properties were typical for the insulin receptor from target tissues for insulin action. The insulin receptor of the normal human brain showed the altered electrophoretic mobility and lack of neuraminidase sensitivity of its alpha-subunit previously reported for the rat brain receptor. There was no difference, however, in the functions of the receptor subunits (binding, phosphorylation) from the normal brain tissue and the eight human gliomal tumors. Since the glial elements compose a majority of the brain cells, the "normal" structure and function of their insulin receptor might provide a key to understanding the role of insulin in the carbohydrate metabolism of the human central nervous system.

Insulin receptor degradation is accelerated in cultured lymphocytes from patients with genetic syndromes of extreme insulin resistance.

Previous studies of the insulin receptor in disease states have utilized primarily techniques of equilibrium binding and, to a limited extent structural, analysis. Though techniques have been developed to study receptor degradation in normal cells, they have not been applied to disease states. In the present study we have examined insulin receptor degradation rate in B lymphocytes that were obtained from peripheral blood of normal subjects and patients with several syndromes of extreme insulin resistance. B lymphocytes were established in culture from each patient's peripheral cells by transformation with Epstein-Barr virus. The insulin receptors were surface labeled using Na125I/lactoperoxidase and the cells were returned to incubate in growth media. After varying periods of incubation, aliquots of cells were solubilized and the cell content of labeled receptor subunits were measured by immunoprecipitation with anti-receptor antibodies and NaDodSO4/polyacrylamide gel electrophoresis. The fall in 125I-insulin receptor content approximated a single exponential and was quantitated as receptor subunit half-life (t1/2). In cell lines from four patients in whom the number of insulin receptors was reduced by greater than 90%, the rate of receptor loss was greater than normal (t1/2 equals 3.8 +/- 0.9 h vs. 6.5 +/- 1.2 h; mean +/- SD, P less than 0.01). However, a similar acceleration in receptor degradation was seen in cells from five patients with extreme insulin resistance but low-normal insulin receptor concentration (t1/2 equals 4.4 +/- 0.9 h). This group included cells from one patient with a qualitatively abnormal receptor. Thus, all the patients with genetic syndromes of insulin resistance had accelerated receptor degradation, regardless of their receptor concentration. By contrast, insulin receptors on cultured lymphocytes that were obtained from patients with extreme insulin resistance secondary to autoantibodies to the insulin receptor had normal receptor degradation (t1/2 equals 6.1 +/- 1.9 h). We conclude that (a) accelerated insulin receptor degradation is an additional feature of cells from patients with genetic forms of insulin resistance; (b) that accelerated insulin receptor degradation may explain the low-normal receptor concentrations that were seen in some patients with extreme insulin resistance; and (c) that accelerated degradation does not explain the decreased receptor concentration in patients with very low insulin receptor binding and, therefore, by inference, a defect in receptor synthesis must be present in this subgroup.

Outcomes of pregnancies affected by impaired glucose tolerance.

OBJECTIVE: Gestational diabetes mellitus (GDM) is associated with an increase in both maternal and neonatal morbidity. There remains uncertainty, however, about the diagnostic criteria for GDM. We compared pregnancy outcomes across three groups of women, with the aim of establishing a threshold for diagnosis of GDM at our institution. METHODS: Women with a glucose tolerance test (GTT) were identified on the hospital's pathology database. Those women with a singleton pregnancy, in whom a GTT had demonstrated a fasting value /=7.8mmol/L and who confined /=5.5mmol/L and/or 2h >/=7.8mmol/L on 75g GTT.

An alteration in apparent molecular weight of the insulin receptor from the human monocyte cell line U-937.

We have studied the structure of the insulin receptor from a human cultured monocyte cell line, U-937. The receptor is composed of alpha and beta subunits as seen in other insulin receptors, but these subunits are of greater apparent molecular weight (alpha 150,000 and beta 102,000) than in typical insulin receptors. Despite this, the U-937 insulin receptor appears to function normally. The alpha subunit binds insulin and the beta subunit is phosphorylated in response to insulin stimulation. Both subunits are expressed in the plasma membrane. Insulin binding isotherms are similar to those seen in IM-9 lymphocytes. Thus, the insulin receptor from U-937 monocytes appears functionally normal despite alterations in molecular weight of the subunits.

Downregulation occurs normally in cultured Epstein-Barr virus-transformed lymphocytes from patients with extreme insulin resistance. Discrepancy between downregulation in vivo and in vitro.

Levels of fasting plasma insulin are generally inversely correlated with 125I-insulin binding to circulating blood cells. In disease states associated with hyperinsulinemia (e.g., obesity and non-insulin-dependent diabetes mellitus), 125I-insulin binding is usually low. In contrast, 125I-insulin binding to circulating cells may be normal in patients with certain forms of extreme insulin resistance despite marked hyperinsulinemia. To explain this paradox, it has been proposed that postbinding defects in insulin action may give rise to defects in downregulation. We have employed cultured Epstein-Barr virus (EBV)-transformed lymphocytes from eight patients with extreme insulin resistance to address the question of whether there is a defect in the downregulation process in vitro. In this cell type, insulin leads to a decrease in the number of insulin receptors on the cell surface by accelerating the rate of degradation of insulin receptors. We could not detect any abnormality in in vitro down-regulation with cultured EBV-transformed lymphocytes from insulin-resistant patients. The apparent discrepancy between the in vivo and in vitro studies raises the possibility that some factor in the patient's internal milieu may prevent insulin-induced downregulation. An alternative possible explanation might be that the mechanism of downregulation in vitro differs from the mechanism whereby receptor number is regulated in vivo in insulin's target cells.

Binding of insulin to its receptor impairs recognition by monoclonal anti-insulin antibodies.

The interaction between insulin and its receptor was investigated using both monoclonal and polyclonal anti-insulin antibodies. After covalent cross-linking of 125I-insulin to the insulin receptor on cultured human lymphocytes (IM-9 cells) using disuccinimidyl suberate, we inquired whether the insulin-receptor complex could be immunoprecipitated with anti-insulin antibodies. While a polyclonal guinea pig anti-insulin antiserum succeeded in immunoprecipitating receptor-bound 125I-insulin, binding to the receptor decreased the avidity of the antiserum for the insulin moiety by a factor of approximately 1000-fold. Sixteen distinct monoclonal murine anti-insulin antibodies were employed to immunoprecipitate receptor-bound 125I-insulin. Of these 16 monoclonal antibodies, only one (antibody 5.9F4) could be shown to recognize receptor-bound 125I-insulin. Moreover, even with antibody 5.9F4, binding of 125I-insulin to its receptor reduced the affinity of the antibody by a factor of 10- to 100-fold. These data strongly suggest that, when insulin binds to its receptor, the majority of the insulin molecule is unavailable for binding by anti-insulin antibodies. It seems likely that the hormone binding site on the receptor may be very large, thereby allowing for sequestration of the majority of the insulin molecule with relatively little of the hormone remaining exposed.

The relationship among homocysteine, creatinine clearance, and albuminuria in patients with type 2 diabetes.

OBJECTIVE: Although it is accepted that elevated plasma homocysteine (tHcy) levels occur in end-stage renal disease and type 2 diabetes, the changes with milder renal dysfunction (e.g., microalbuminuria) are less clearly established. This study explores the relationship among tHcy, creatinine clearance (Ccr), and albumin excretion rate (AER) in a population with type 2 diabetes. RESEARCH DESIGN AND METHODS: A total of 260 patients with type 2 diabetes were screened in our outpatient clinic during 10 months. Fasting blood samples were collected, and AER was calculated from an overnight timed urine sample. Ccr was calculated using the Cockroft-Gault formula. RESULTS: A total of 198 subjects (76%) had normoalbuminuria (<20 microg/min), 50 subjects (19%) had microalbuminuria (20-200 microg/min), and 12 subjects (5%) had macroalbuminuria (>or=200 microg/min). Those with microalbuminuria had higher levels of tHcy than those with normoalbuminuria (13.2 +/- 7.8 vs. 11.3 +/- 4.6 micromol/l, P < 0.05). Patients were then subdivided based on low Ccr (<80 ml x min(-1) x 1.73 m(-2)) and normal Ccr (>or=80 x min(-1) x 1.73 m(-2)). None of the patients with macroalbuminuria had normal Ccr. In those with normoalbuminuria, tHcy levels were higher than in those with low Ccr than in those with normal Ccr (12.0 +/- 4.6 vs. 10.0 +/- 4.4 micromol/l, P < 0.01). The same was found for those with microalbuminuria (low Ccr versus normal Ccr: 14.6 +/- 9.0 vs. 10.2 +/- 2.8 micromol/l, P < 0.02). For normal Ccr, tHcy was similar irrespective of AER (normoalbuminuria versus microalbuminuria: 10.0 +/- 4.4 vs. 10.2 +/- 2.8 micromol/l, NS). For low Ccr, tHcy was higher in those with microalbuminuria versus normoalbuminuria (14.6 +/- 9.0 vs. 12.0 +/- 4.6 micromol/l, P = 0.01). Using multivariate regression, Ccr, but neither AER nor the presence of albuminuria, was an independent predictor of tHcy. CONCLUSIONS: These data strongly suggest that in patients with type 2 diabetes, the relationship between plasma tHcy and AER is largely due to associated changes in renal function, as defined by Ccr.

Mortality associated with primary hyperparathyroidism.

561 patients with primary hyperparathyroidism were followed between 1961 and 1994. Relative survival was compared to that of the Australian population studied during the same time interval. Mortality was significantly greater in the hyperparathyroid population (P<0.001). Mortality was not greater in the patients with serum calcium levels >3.00 mmol/L compared to those with a serum calcium levels <3.00 mmol/L. 113 patients did not have parathyroid surgery. Their relative survival was not significantly different from those who had surgery but their mean serum calcium and parathyroid hormone (PTH) levels were significantly lower than those who had surgery. A re-analysis of the 453 patients followed between 1972 and 2011 was carried out and a 20-year survival analysis made of those diagnosed between 1972 and 1981 and those diagnosed between 1982 and 1991. The latter group had significantly worse relative mortality than the former group (P<0.001) but was significantly older at the time of diagnosis (56.94 ± 14.83 vs 52.01 ± 13.58, P<0.001). The serum calcium and serum PTH levels were not significantly different between these two groups.

Longitudinal changes in forearm bone mineral content in primary hyperparathyroidism.

Forearm bone mineral content was measured in 28 patients with primary hyperparathyroidism before and 1 year after successful parathyroidectomy. The forearm bone mineral content rose from a mean value of 1.068 to 1.092 g/cm (P less than 0.05, paired t-test). Those patients with the lower initial values had the largest rise. In an additional study, the forearm bone mineral content was measured in 10 women over the age of 40 years (mean age 58.6 +/- 7.9SD years) with hyperparathyroidism before and for 2 years after successful parathyroidectomy and compared with the forearm bone mineral content measured over 2 years in 12 women (mean age 56.3 +/- 5.5SD years) with continuing hyperparathyroidism and with the forearm bone mineral content of 12 eucalcemic control women (mean age 58.8 +/- 8.2SD years), also measured over 2 years. The parathyroidectomized group gained bone, whereas the ongoing hyperparathyroid group and the eucalcemic control group lost bone. The difference between the parathyroidectomized group and the ongoing hyperparathyroid group was significant after 2 years (P less than 0.05). The percentage loss of forearm bone mineral in the eucalcemic control subjects was not significantly different from the percentage loss of forearm bone mineral in the ongoing hyperparathyroid group, although the initial mean bone mineral content in the eucalcemic group was significantly higher than in the ongoing hyperparathyroid group, suggesting that a possible determinant of bone mineral loss in women in this age group is the initial bone mineral content.

Insulin promotes growth of the cultured rat osteosarcoma cell line UMR-106-01: an osteoblast-like cell.

The rat osteogenic sarcoma subclone UMR-106-01 is a cell type with osteoblast-like properties. This cell line has been shown to process specific receptors for insulin and insulin-like growth factor I (IGF-I), but not IGF-II. Insulin at physiological concentrations (1-5 ng/ml) in serum-free medium can maintain cell growth, as assessed by protein accumulation, thymidine uptake, and an increase in cell number. IGF-I is less potent than insulin, but, based on relative binding affinities for the insulin receptor, possibly acts via its own receptor. Insulin also enhances PTH-stimulated cAMP accumulation in these cells both by increasing cell number and an effect independent of cell number. Insulin may have a role in bone homeostasis.

The insulin-like growth factor-II (IGF II) receptor from rat brain is of lower apparent molecular weight than the IGF II receptor from rat liver.

The binding subunits of the insulin and insulin-like growth factor-I (IGF I) receptors from rat brain are of lower molecular weight than the corresponding receptor in rat liver, possibly due to variations in sialic acid content. We have compared the IGF II receptor from rat brain and rat liver. The brain receptor is of smaller apparent mol wt (about 10 K) on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This size difference is independent of ligand binding as it persists in iodinated and specifically immunoprecipitated receptors. From studies of wheat germ agglutinin binding and the effect of neuraminidase on receptor mobility, we conclude that this difference is not simply due to variations in sialic acid content. Treatment with endoglycosidase F results in reduction in the molecular size of both liver and brain receptors and after this treatment the aglycoreceptors are of similar size. We conclude that in rat brain tissue the IGF II receptor like the binding subunits of the insulin and IGF I receptors is of lower molecular size than the corresponding receptors in rat liver. This difference is due to differences in N-linked glycosylation.

Polypeptide hormone receptor phosphorylation: is there a role in receptor-mediated endocytosis of human growth hormone?

To determine whether receptor phosphorylation is a critical step in the internalization of polypeptide hormones and their receptors, we have studied a model system wherein insulin stimulates phosphorylation of its receptor and is also internalized. Using insulin as a positive control, we found that it stimulated a partially purified plasma membrane preparation of IM-9 lymphocytes to autophosphorylate its receptor and to catalyze the phosphorylation of a tyrosine-containing substrate. The human GH (hGH) receptor of the IM-9 lymphocytes, when coupled to [125I]iodo-hGH, migrated as a 140,000-dalton protein on polyacrylamide gel electrophoresis. This protein, in contrast to the insulin receptor, was not phosphorylated by the addition of hGH, nor did hGH stimulate this preparation to phosphorylate the tyrosine-containing substrate poly-(GluNa,Tyr)4:1, casein, or histone f2b under a variety of conditions. We conclude that receptor phosphorylation is not a critical intermediate in the receptor-mediated endocytosis of hGH and probably other polypeptide hormones and growth factors.

Insulin bound to the insulin receptor of IM-9 lymphocytes is more accessible to antiinsulin antibodies after treatment with dithiothreitol: bound insulin is deeply buried in the receptor-binding site.

Binding of insulin to its receptor followed by covalent cross-linking with disuccinimidyl suberate (DSS) dramatically impairs the ability of antiinsulin antibodies (both polyclonal and monoclonal) to bind to the insulin moiety. We have used dithiotheitol, which has major effects on the oligomeric structure of the insulin receptor, to determine if this decreased antibody recognition is due to alteration in the conformation of insulin itself or to steric factors. Treatment of the covalently cross-linked insulin-receptor complex with dithiothreitol (DTT) increased the ability of the polyclonal and two monoclonal antiinsulin antibodies to immunoprecipitate the insulin-receptor complex. This treatment decreased immunoprecipitation by antireceptor antibodies. The effect of DTT may have been due to a reversal of either a binding-induced conformational change in the insulin moiety or an alteration in the conformation of the insulin-receptor complex, thereby decreasing steric hindrance. In an effort to choose between these two possible explanations, we prepared a biotinylated derivative of insulin which was cross-linked to the receptor. Since the biotin moiety is relatively rigid, it seemed improbable that binding to the receptor would alter the conformation of the epitope recognized by antibiotin antibodies and that the change would be reversed by DTT. Treatment of the cross-linked biotin-insulin-receptor complex with DTT did increase the ability of both antiinsulin and antibiotin antibodies to immunoprecipitate the cross-linked receptor complex. Identification of the cross-linked receptor on reduced sodium dodecyl sulfate-polyacrylamide gels confirms that the DTT treatment alters the distribution of the oligomeric forms of the receptor. These studies favor the hypothesis that when bound to its receptor, most of the insulin molecule is sequestered within the receptor-binding site such that there is steric hindrance to the approach of antiinsulin antibodies. Moreover, DTT alters the conformation of the cross-linked insulin-receptor complex so as to decrease this steric hindrance.