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D H Lockwood

Publications and source records attributed to D H Lockwood.

At least 19 recordsLinked to original sources
PubMed1991

Mapping of carbohydrate sites on the human insulin receptor.

Prior to investigating the role of individual glycosylation sites in insulin receptor function, we are mapping the sites of glycosylation in the receptor. We report here a generally applicable methodology for the isolation and identification of glycosylation sites in cell surface glycoproteins. Human insulin receptors were labeled with [3H]-sugars using a CHO cell line transfected with the human receptor cDNA. Labelled receptors were mixed with receptors purified from human placental membranes and tryptic peptides prepared. Peptides were fractionated by gel filtration chromatography to limit the number of non-glycopeptides present. Peptides were then separated by reverse phase HPLC and glycopeptides identified by scintillation counting. Using this technique we have shown the insulin receptor to be glycosylated at Asn 397 and Asn 881. This increase the known number of occupied glycosylation sites to five.

Amino Acid Sequence↗
PubMed1991

Intermolecular phosphorylation of insulin receptor as possible mechanism for amplification of binding signal.

Clustering of cell-surface insulin receptors has led to the speculation that intermolecular phosphorylation of unoccupied receptors catalyzed by ligand-occupied receptors within the cluster could be a mechanism by which the insulin-binding signal is amplified. We examined whether insulin receptors can be phosphorylated by an intermolecular mechanism. In this study, we used highly purified insulin receptors isolated from rat liver plasma membranes and human placental membranes. Rat liver insulin receptors were "activated" by incubation with 10 nM insulin in the presence of ATP. Subsequent to removal of insulin by immunodepletion, these receptors were used as an enzyme source to study phosphorylation of unphosphorylated "substrate" human receptors. Initially, we found no evidence that the addition of activated rat receptors increased phosphorylation of human receptors, when assessed by immunoprecipitation with a human-specific monoclonal antibody. To examine the possibility that these negative results were due to insufficient receptor concentration, activated human receptors were mixed with unphosphorylated substrate receptors at concentrations up to 60 micrograms/ml. In this study, we found that addition of activated receptors resulted in increased phosphorylation of the substrate receptors at the highest concentrations employed. These are the first data indicating that insulin receptors per se are capable of intermolecular phosphorylation. In vivo, this could be the initial step in amplifying the insulin-binding signal.

Animals↗
PubMed1990

A guide to fragile sites on human chromosomes.

This is a guide to 107 fragile sites, all those considered at the most recent International Workshop on Human Gene Mapping, HGM 9.5, held in 1988. The chromosome band locations of all 107 fragile sites are given, together with their gene symbols, frequency, mode of induction, and status. The majority of these fragile sites are common ones induced to expression by aphidicolin. Fragile sites are nonrandomly distributed within the genome. Chromosome 3 is especially short of known fragile sites. Chromosome 21, the chromosome triplicated in Down syndrome, has no known fragile sites.

Aphidicolin↗
PubMed1989

Not all chromosome imbalance resulting from the 11q;22q translocation is due to 3:1 segregation in first meiosis.

The constitutional translocation between chromosomes 11 and 22 [t(11;22)(q23.3;q11.2)] is one of the best known rearrangements in the human genome. Hitherto only one type of unbalanced karyotype, namely 47,XX or XY, +der(22) t(11;22)(q23.3;q11.2) was found among offspring of the translocation carriers. This result is the product of a 3:1 segregation at meiosis. We report an alternative unbalanced karyotype. The proband's karyotype is 47,XY,t(11;22)(q23.3;q11.2), +der(22)t(11;22)(q23.3;q11.2)pat. This finding cannot be due to nondisjunction in first meiosis of the translocation carrier.

Chromosome Banding↗
PubMed1989

Alterations of glucose transporter systems in insulin-resistant uremic rats.

To further define the cellular alteration(s) involved in the impaired glucose transport associated with chronic uremia, we examined the concentration and translocation of glucose transport systems in adipocytes isolated from partially nephrectomized uremic rats. Uremic animals, compared with matched controls, had increased blood urea nitrogen and serum insulin, whereas serum glucose was unchanged. In agreement with previous work, 125I-insulin binding to its receptor was unaltered and transport of 2-deoxy-D-glucose was decreased in both the absence (basal) and presence of a maximal (7 nM) insulin concentration by 44 and 35%, respectively. To assess the movement and concentration of glucose transport systems in various membrane fractions prepared from basal and insulin-treated (20 nM) uremic fat cells, the technique of D-glucose-inhibitable cytochalasin B binding was utilized. In plasma membranes isolated from these cells the concentration of glucose transporters was decreased by 16 (P less than 0.01) and 30% (P less than 0.005) in basal and insulin-treated cells, respectively. Concomitantly, microsomal membranes prepared from uremic cells treated in the absence and presence of insulin had a 28 (P less than 0.01) and 15% (P less than 0.05) decrease in concentration of glucose transport systems, respectively. Additionally, glucose transporter concentration was significantly decreased by 17% (P less than 0.025) in total membranes prepared from uremic cells. Thus, impairment of glucose transport in uremic fat cells can be attributed to a postbinding defect that, at least in part, results from a decrease in the total concentration of glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1989

In vitro effects of sulfonylurea on glucose transport and translocation of glucose transporters in adipocytes from streptozocin-induced diabetic rats.

The in vitro effects of the sulfonylurea glyburide on insulin binding and action were compared in adipocytes from control and nonketotic streptozocin-induced diabetic rats. Adipose tissue from control and diabetic animals was maintained in the absence or presence of 2 micrograms/ml glyburide for 20 h. Insulin binding and insulin-stimulated glucose transport were examined in adipocytes prepared from this tissue. As expected, insulin binding was increased in adipocytes from diabetic animals. Exposure of tissue to glyburide did not influence insulin binding in either control or diabetic cells. Glucose transport activity of diabetic cells, assessed with 2-deoxyglucose, was decreased 30-40% in both the absence (basal) and presence of insulin compared with controls. Glyburide potentiated insulin's effects in both control (15-20%) and diabetic (30-40%) adipocytes. As a result, glucose transport activity in glyburide-treated diabetic cells was restored to a level similar to that of control cells not exposed to the drug. The mechanism by which glyburide potentiated glucose transport activity was examined with the D-glucose-displaceable cytochalasin B-binding technique to measure glucose-transporter concentration in membranes prepared from control and diabetic adipocytes exposed to the drug. Adipocytes from this model of diabetes are known to have a decreased cellular content of glucose transporters. The concentration of glucose transporters was decreased by 31% in plasma membranes from insulin-treated diabetic cells. There were corresponding decreases in diabetic microsomal and total membrane fractions. There was also a 40% decrease in the translocation of transporters from the microsomes to the plasma membrane in response to insulin in diabetic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1988

Intact adipocyte insulin-receptor phosphorylation and in vitro tyrosine kinase activity in animal models of insulin resistance.

We evaluated the possibility that impaired insulin-receptor kinase activity contributes to insulin resistance by examining in vitro receptor tyrosine kinase activity and in situ receptor phosphorylation in four models of insulin resistance. Adipocytes from streptozocin-induced nonketotic diabetic (STZ-D), glucocorticoid-treated, fasted, and chronically uremic rats showed reduced basal and maximally insulin-stimulated 2-deoxy-D-glucose transport compared with matched controls. Adipocytes from these models were also resistant to stimulation of hexose transport by hydrogen peroxide, a postbinding insulin mimicker. Changes in the number of insulin receptors per cell could not account for these alterations in transport. Cell surface 125I-labeled insulin binding was 142% of control in STZ-D and 129% with fasting and unchanged in glucocorticoid excess and chronic uremia. Insulin-stimulated tyrosine kinase was measured by means of a synthetic substrate, Glu80Tyr20. Partially purified receptors from these resistant models had unaltered kinase activity when normalized to soluble 125I-insulin binding. In situ stimulation of receptor phosphorylation by 7 and 100 nM insulin was determined after equilibration of adipocytes with 32PO4. Compared with matched controls, these intact cells, from all four resistant models, had insulin-stimulated receptor phosphorylation that was unchanged per unit of cell surface binding. Similar to results with insulin, hydrogen peroxide stimulation of in situ receptor phosphorylation was unchanged in each model. Thus, both in vitro and in situ measures of receptor phosphorylation suggest that the cellular alterations leading to insulin resistance in these adipocytes resides beyond phosphorylation of the insulin receptor.

Adipose Tissue↗
PubMed1988

The use of subchromosome-length unique band sequences in the analysis of prophase chromosomes.

Using human prophase chromosome ideograms at the 850-band stage, we previously demonstrated that the 24 prophase ideograms can be divided into a set of 94 unique band sequences, each having a recognizable banding pattern distinct from other nonhomologous chromosome portions. Using actual prophase mitotic cells in this study, we analyzed the p arm of chromosome 11 and of chromosomes 16-22 and characterized a similar set of unique band sequences on actual chromosomes. This set of unique band sequences, a statistical comparison scheme, and image-processing techniques outlined in the present report can be used to identify and distinguish banding patterns of these chromosomes and to determine band pattern abnormalities.

Chromosome Aberrations↗
PubMed1988

The safety and efficacy of a controlled low-energy ('very-low-calorie') diet in the treatment of non-insulin-dependent diabetes and obesity.

We evaluated the safety and efficacy of a highly supplemented controlled low-energy (1764 kJ [420 kcal]) diet in the treatment of non-insulin-dependent diabetes and obesity. Six obese, diabetic women ranging from 143% to 297% of ideal body weight were studied in a metabolic ward for 48 days. The subjects ingested a weight-maintenance diet during an eight-day control period followed by 40 days of an experimental diet containing 1764 kJ (420 kcal) of a mixture of protein (43% of energy intake), carbohydrates (51%), and fat (6%), supplemented with minerals, trace elements, and vitamins. The subjects were monitored for balances of nitrogen and minerals, as well as for the appearance of cardiac arrhythmias by 24-hour electrocardiographic recordings. Weight loss was rapid and sustained and averaged 10.1% +/- 0.8% over 40 days. Fasting plasma glucose levels declined from 16.2 +/- 1.9 mmol/L (293 +/- 36 mg/dL) to 6.9 +/- 0.8 mmol/L (126 +/- 16 mg/dL) by day 35. Similarly, hemoglobin A1c levels fell from 0.11 +/- 0.009 (11.2% +/- 0.9%) to 0.8 +/- 0.001 (8.2% +/- 1.1%). Urinary C-peptide levels declined from 62.2 +/- 15.6 nmol/48 h to 20.0 +/- 5.9 nmol/48 h by days 39 to 40 and paralleled the decline in plasma glucose values, the majority of which occurred in the first seven days. Concentrations of serum cholesterol and triglycerides decreased. Balances for nitrogen, potassium, and magnesium were negative at -1.7 g/24 h, -2.2 mEq/24 h, and -2.9 mg/dL, respectively. Blood pressure decreased without orthostasis. Resting metabolic rate fell a mean of 18% but remained within normal limits. Triiodothyronine levels also declined. Twenty-four-hour ambulatory electrocardiographic readings disclosed no significant bradyarrhythmia or tachyarrhythmia for any patient. These studies, based on a limited number of subjects, demonstrate that a highly supplemented controlled low-energy diet is a safe and efficacious treatment for diabetes and obesity, leading to significant decreases in weight, blood pressure, and levels of plasma glucose and plasma lipids. Such diets may be the optimal initial treatment of moderate to markedly obese patients with non-insulin-dependent diabetes.

Adult↗
PubMed1987

Effect of chlorpropamide on glucose transport in rat adipocytes in the absence of changes in insulin binding and receptor-associated tyrosine kinase activity.

In an attempt to elucidate the cellular mechanism(s) by which sulfonylureas exert their extrapancreatic hypoglycemic effects, various parameters of insulin action were examined in vitro, using rat adipocytes maintained in a biochemically defined medium. Cells were maintained for 20 hours in the absence or presence of 175 micrograms/mL chlorpropamide and insulin binding, hexose transport, glucose metabolism, and insulin receptor tyrosine kinase activity were compared. Chlorpropamide treatment had no effect on insulin binding, altering neither receptor number nor affinity. However, the sulfonylurea did enhance 2-deoxyglucose transport in both the absence (17%, P less than .01) and presence (20%, P less than .01) of insulin. Furthermore, glucose metabolism as measured by the conversion of glucose (0.2 mmol/L) to CO2 and total lipids was also significantly increased by chlorpropamide treatment in both the absence (30%, P less than .01) and presence (31%, P less than .05) of insulin. Potentiation of insulin-stimulated transport or metabolism was not explained by an increase in the basal state alone because the incremental responses to 40 ng/mL insulin were potentiated by 19% (P less than .01) and 25% (P less than .05), respectively. Activity of the insulin receptor kinase was unchanged as evaluated by autophosphorylation of partially purified receptors, phosphorylation of an artificial substrate and by phosphorylation of the receptor in situ. These studies demonstrate that the sulfonylurea, chlorpropamide, stimulates glucose transport and potentiates insulin's effect on this process by acting at a site(s) beyond insulin receptor binding and phosphorylation.

Adipose Tissue↗
PubMed1987

Role of insulin receptor phosphorylation in the insulinomimetic effects of hydrogen peroxide.

The oxidant H2O2 has many insulin-like effects in rat adipocytes. To determine whether these effects could be mediated by the tyrosine kinase activity of the insulin receptor, the ability of H2O2 to stimulate receptor phosphorylation in intact adipocytes and partially purified insulin receptors has been examined. Phosphorylation of the beta subunit of the insulin receptor was increased approximately 2-fold by treatment of intact cells with 3 mM H2O2, a concentration that maximally stimulates 2-deoxyglucose uptake. Stimulation of receptor phosphorylation was rapid, reaching maximal levels within 5 min, and preceded activation of glucose transport. Phosphoamino acid analysis of insulin receptors from H2O2-treated adipocytes showed that 32P incorporation into phosphotyrosine and phosphoserine residues of the beta subunit was enhanced. Furthermore, partially purified receptors from H2O2-treated cells exhibit increased tyrosine kinase activity, as measured by phosphorylation of the peptide Glu80Tyr20. In contrast, the direct addition of H2O2 to partially purified insulin receptors did not stimulate tyrosine kinase activity or insulin receptor autophosphorylation. This was not due to breakdown of H2O2 or oxidation of ATP or the required divalent cations. To define the factors involved in H2O2's effect, we have examined receptor phosphorylation in fat cell homogenates and purified plasma membranes. Although insulin stimulated receptor phosphorylation in both of these systems, H2O2 was only effective in the cell homogenates. These data demonstrate that, under certain conditions, H2O2 stimulates insulin receptor phosphorylation and tyrosine kinase activity, suggesting that the insulin-like effects of H2O2 may be mediated by stimulation of insulin receptor phosphorylation. This does not appear to be a direct effect of H2O2 on the insulin receptor and requires nonplasma membrane cellular constituents.

Adipose Tissue↗
PubMed1986

The role of cell surface sialic acid in insulin receptor function and insulin action.

Removal of cell surface sialic acid from adipocytes with neuraminidase inhibits insulin action. Here, we have examined the effects of mild neuraminidase treatment (5 milliunits/ml, 12 degrees C, 15 min) on insulin receptor structure and function. Neuraminidase treatment sufficient to cause greater than 90% loss of insulin stimulatable lipogenesis had no effect on 125I-insulin binding, tyrosine kinase activity of partially purified insulin receptors, insulin receptor phosphorylation in intact cells, or insulin-induced receptor internalization. However, recycling of the insulin receptor to the plasma membrane was inhibited by 50%. Recycled receptors in neuraminidase-treated cells were unable to mediate insulin action in contrast to recycled receptors from non-neuraminidase-treated cells. Furthermore, when insulin receptors were protected from exposure to neuraminidase, by inducing receptor internalization prior to neuraminidase treatment, the cells were still unable to respond to insulin. Analysis of the alpha and beta subunits of the receptor from neuraminidase-treated cells, affinity-labeled with 125I-insulin, or labeled by autophosphorylation, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis failed to indicate any changes in the holoreceptor or the individual subunits. This suggests there was no detectable release of sialic acid from the receptor. From this data we conclude that loss of sialic acid from nonreceptor glycoconjugates leads to loss of insulin action and inhibition of receptor recycling. The insulin receptor does not appear to be involved in this inhibitory effect. These findings suggest that an uncharacterized plasma membrane glycoprotein is essential in transmitting the "signal" of insulin binding to the cellular effector system.

Adipose Tissue↗
PubMed1986

Effects of metformin on insulin receptor tyrosine kinase activity in rat adipocytes.

The cellular mechanism(s) by which the biguanide, metformin, exerts its antihyperglycaemic effect was investigated. Rat adipocytes were either treated acutely (2 h) or maintained in a biochemically defined medium (20 h) in the presence or absence of metformin (1 X 10(-4) mol/l). Exposure to the drug resulted in a significant enhancement (p less than 0.01) of hexose transport in both the absence (basal) and presence of insulin. Stimulation of transport was not explained by the increase in the basal state alone, since the incremental response to maximally effective concentrations of insulin was significantly enhanced p less than 0.025. Insulin-receptor tyrosine kinase activity was examined under the same experimental conditions. Activity of the kinase was unaltered as evaluated by phosphorylation of an artificial substrate and by phosphorylation of the receptor in situ. Furthermore, in this investigation neither insulin receptor number nor affinity was changed in adipose tissue treated with metformin. These studies indicate that metformin potentiates the effect of insulin on glucose transport at a site(s) beyond insulin receptor binding and phosphorylation.

Adipose Tissue↗
PubMed1986

Prophase chromosome unique band sequences: definition and utilization.

Extensive experience with the analysis of human prophase chromosomes and studies into the complexity of prophase banding patterns have suggested that at least some prophase chromosomal segments can be accurately identified and characterized independently of the morphology of the chromosome as a whole. The feasibility of identifying and analyzing specified prophase chromosome segments was thus investigated as an alternative approach to prophase chromosome analysis based on whole-chromosome recognition. Through the use of prophase idiograms at the 850-band stage (Francke, 1981) and a systematic comparison system, we have demonstrated that it is possible to divide the 24 human prophase idiograms into a set of 94 unique band sequences, each of which has a banding pattern that is recognizable and distinct from any other nonhomologous chromosome portion. The use of a unique band sequence approach in prophase chromosome analysis is expected to increase efficiency and sensitivity through more effective use of available banding information.

Chromosome Banding↗
PubMed1985

Insulin resistance: receptor and post-binding defects in human obesity and non-insulin-dependent diabetes mellitus.

Insulin resistance is a prominent feature of three clinical conditions: obesity, impaired glucose tolerance, and non-insulin-dependent (type II) diabetes mellitus. Numerous studies over the past 15 years have provided a better understanding, from both a clinical and cellular standpoint, of the pathophysiology of these insulin-resistant states as well as of insulin action. In addition, it has recently been recognized that correction of glucose intolerance leads to an improvement in insulin secretion and a reduction in insulin resistance. Examination of the most recent data suggests that the basis for insulin resistance in these common clinical disorders is often multifactorial. In uncomplicated obesity, the cellular alterations responsible for insulin resistance appear to be at the level of the hepatic insulin receptor and in post-binding processes in peripheral target tissues. In type II diabetes, a post-binding defect(s) in peripheral tissues appears to be the primary lesion. In humans, many of the factors that mediate the changes leading to insulin resistance are still unknown and are the object of current investigations.

Adipose Tissue↗