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Biomedical subjects

M Landt

Publications and source records attributed to M Landt.

At least 55 records · Page 3Linked to original sources
PubMed1996

Muscarinic activation of Ca2+/calmodulin-dependent protein kinase II in pancreatic islets. Temporal dissociation of kinase activation and insulin secretion.

We have demonstrated previously that glucose activates the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in isolated rat pancreatic islets in a manner consistent with a role of this enzyme in the regulation of insulin secretion [Wenham, Landt and Easom (1994) J. Biol. Chem. 269, 4947-4952]. In the current study, the muscarinic agonist, carbachol, has been shown to induce the conversion of CaM kinase II into a Ca(2+)-independent, autonomous form indicative of its activation. Maximal activation (2-fold) was achieved by 15 s, followed by a rapid return to basal levels by 1 min. This response was primarily the result of the mobilization of Ca2+ from intracellular stores since it was not affected by a concentration (20 microM) of verapamil that completely prevented the activation of CaM kinase II by glucose. Surprisingly, carbachol added prior to, or simultaneously with, glucose attenuated nutrient activation of CaM kinase II. This effect was mimicked by cholecystokinin-8 (CCK-8) and thapsigargin, suggesting its mediation by phospholipase C and the mobilization of intracellular Ca2+. In contrast, carbachol, CCK-8 and thapsigargin markedly potentiated glucose (12 mM)-induced insulin secretion. These results suggest that CaM kinase II activation can be temporally dissociated from insulin secretion but do not exclude the potential dependence of insulin exocytosis on CaM kinase II-mediated protein phosphorylation.

Animals↗
PubMed1996

Serum leptin levels in women with anorexia nervosa.

Leptin is a protein encoded by the ob gene that is expressed in adipocytes and regulates eating behavior via central neuroendocrine mechanisms. Serum leptin levels have been shown to correlate with weight and percent body fat in normal and obese individuals; however, it is not known whether the regulation of leptin is normal below a critical threshold of body fat in chronic undernutrition. We investigated serum leptin levels in 22 women, aged 23 +/- 4 yr, with anorexia nervosa. Duration of disease, weight, BMI, percent body fat, and serum leptin levels were determined for each patient. Nutritional status was assessed further by caloric intake and measurement of insulin and insulin-like growth factor I (IGF-I) levels. Twenty-three healthy women, aged 23 +/- 4 yr, taking no medications, with normal menstrual function and body mass index (BMI) between 20-26 kg/m2 (mean, 23.7 +/- 1.7 kg/m2), served as a control population for comparison of leptin levels. Subjects with anorexia nervosa were low weight (BMI, 16.3 +/- 1.6 kg/m2; normal, 20-26 kg/m2) and exhibited a striking reduction in percent body fat (7 +/- 2%; normal, 20-30%). The mean serum leptin level was significantly decreased in subjects with anorexia nervosa compared with that in age- and sex-matched controls of normal body weight (5.6 +/- 3.7 vs. 19.1 +/- 8.1 ng/mL; P < 0.0001). Serum leptin levels were correlated highly with weight, as expressed either BMI (r = 0.66; P = 0.002) or percent ideal body weight (r = 0.68; P = 0.0005), body fat (r = 0.70; P = 0.0003), and IGF-I (r = 0.64; P = 0.001), but not with caloric intake or serum levels of estradiol or insulin in subjects with anorexia nervosa. The correlation between leptin and body fat was linear, with progressively lower, but detectable, leptin levels measured even in patients with less than 5% body fat, but was not significant when the effects of weight were taken into account. In contrast, the correlation between leptin and IGF-I remained significant when the effects of weight, body fat, and caloric intake were taken into account. In normal controls, leptin correlated with BMI (r = 0.55; P = 0.007) and IGF-I (r = 0.44; P < 0.05), but not with fat mass. These data demonstrate that serum leptin levels are reduced in association with low weight and percent body fat in subjects with anorexia nervosa compared to normal controls. Leptin levels correlate highly with weight, percent body fat, and IGF-I in subjects with anorexia nervosa, suggesting that the physiological regulation of leptin is maintained in relation to nutritional status even at an extreme of low weight and body fat.

Adipose Tissue↗
PubMed1996

Serum leptin levels are reduced in response to exercise training, but not hormone replacement therapy, in older women.

The aim of this study was to evaluate the effects of exercise training and hormone replacement therapy (HRT) on serum leptin levels in older women. Previously sedentary, healthy women, aged 60-72 yr, were assigned to control (n = 16), exercise (n = 17), HRT (n = 15), or exercise + HRT (n = 13) groups. Exercise training consisted of a 2-month flexibility-exercise program followed by a 9-month exercise program that included walking, jogging, and stair climbing. HRT consisted of 11 months of continuous conjugated estrogens (0.625 mg/day) and medroxyprogesterone acetate (5 mg/day) for 13 days every third month. Body composition was assessed by dual-energy x-ray absorptiometry, and serum insulin levels were measured in the fasted state and in response to a glucose challenge. Leptin levels were reduced by 23 +/- 25% and 22 +/- 27% (both P < 0.01) in response to exercise and exercise + HRT, respectively. There was no effect of HRT on leptin. Fat mass was the strongest predictor of serum leptin concentration, both before (r = 0.81; P < 0.001) and after (r = 0.85; P < 0.001) the study period, and the change in fat mass in the exercisers was significantly correlated with the change in leptin (r = 0.55; P < 0.01). There did not seem to be an effect of exercise, independent of the reduction in fat mass, on leptin. Insulin levels were significantly correlated with leptin levels, but this was not independent of the association with adiposity. The curvilinear relationship between leptin level and fat mass and the finding that the ratio of leptin mass to fat mass decreased after weight loss suggest that fat cell size is an important determinant of circulating leptin levels.

Adipose Tissue↗
PubMed1996

Plasma leptin and insulin relationships in obese and nonobese humans.

Hyperinsulinemia. is associated with an overexpression of mRNA for the ob protein leptin in rodent models of genetic obesity, and insulin has been reported to directly stimulate leptin mRNA in rat adipocytes. Human obesity is also associated with increased leptin mRNA as well as plasma levels, but there have been no reports of the effect of insulin on leptin secretion. We, therefore, tested the hypothesis that insulin stimulates leptin secretion in humans. Using a newly developed leptin assay, immunoreactive leptin was measured in fasting and postprandial plasma samples from 27 healthy adults and in samples before and during euglycemic-hyperinsulinemic then stepped hypoglycemic (hourly steps at 85, 75, 65, 55, and 45 mg/dl) clamps from 10 healthy subjects and 11 patients with IDDM. Plasma leptin was correlated (r = 0.84, P = 0.0005) with BMI in obese but not nonobese subjects and with fasting (r = 0.75, P = 0.008) but not postprandial plasma insulin levels. (Leptin levels did not change postprandially.) Euglycemic hyperinsulinemia did not alter leptin levels, nor did hyperinsulinemic hypoglycemia. Thus, because circulating leptin levels are not increased during postprandial hyperinsulinemia or during euglycemic (or hypoglycemic) hyperinsulinemia, we conclude that, at least in the short term, insulin does not increase leptin secretion in humans and that hyperleptinemia in obese individuals is not likely the result of hyperinsulinemia.

Adipocytes↗
PubMed1996

Adipose tissue leptin production and plasma leptin kinetics in humans.

Abdominal adipose tissue leptin production was determined in vivo by arteriovenous balance in 14 lean and obese men (mean BMI 27.0 +/- 1.9, range 21.4-45.2). Blood samples were taken simultaneously from an abdominal vein that drains subcutaneous adipose tissue and from a radial artery. Adipose tissue blood flow was measured by xenon washout. Abdominal vein leptin concentrations (mean 8.9 +/- 2.4 ng/ml, range 2.1-36.5 ng/ml) were consistently greater than arterial values (mean 6.6 +/- 1.9 ng/ml, range 1.7-28.2 ng/ml) (P < 0.001). The net rate of abdominal adipose tissue leptin production (mean 3.2 +/- 0.5 ng x 100 g(-1) x min(-1)) correlated directly with percentage body fat (rs = 0.59, P = 0.016). Estimated whole-body leptin production rate (797 +/- 283 ng x person(-1) x min(-1)) correlated directly with percent body fat (rs = 0.93, P < 0.0001) and with regional leptin production (rs = 0.81, P < 0.001). In contrast, the rate of leptin clearance from plasma (mean 1.50 +/- 0.23 ml x kg(-1) x min(-1)) and plasma leptin half-life (mean 24.9 +/- 4.4 min) was unrelated to adiposity (rs = 0.06, P = 0.30; rs = 0.16, P = 0.30, respectively). These results provide direct evidence that leptin is produced by adipose tissue in humans and that the rate of production is directly related to adiposity. A combination of greater leptin production per unit of body fat and increased production from expanded total body fat mass, rather than alterations in leptin clearance, account for the increase in plasma leptin concentrations observed in obese humans.

Abdomen↗
PubMed1996

Relationship between insulin sensitivity and plasma leptin concentration in lean and obese men.

Alterations in the production of or the sensitivity to leptin, the protein encoded by the ob gene, cause obesity and diabetes in rodents. We evaluated the isolated relationship between leptin and insulin sensitivity in lean and obese humans. Three groups of subjects who were carefully matched for either insulin sensitivity (determined by the modified intravenous glucose tolerance test and minimal model analysis) or adiposity (determined by hydrodensitometry) were studied: 1) lean insulin-sensitive men (percentage body fat, 15 +/- 1%); 2) lean insulin-resistant men (percentage body fat, 16 +/- 1%), matched on percentage body fat and fat mass with the lean insulin-sensitive group; and 3) obese insulin-resistant men (percentage body fat, 31 +/- 3), matched on insulin sensitivity with the lean insulin-resistant group. Basal plasma leptin concentrations were significantly lower in the lean insulin-sensitive than in the lean insulin-resistant men (1.90 +/- 0.4 vs. 4.35 +/- 1.21 ng/ml, P < 0.05) despite identical body composition. Plasma leptin in the obese men (9.27 +/- 1.4 ng/ml) was significantly higher than values in the two lean groups (P < 0.01). Marked alterations in plasma glucose and insulin concentrations induced by glucose and tolbutamide injection did not cause any change in plasma leptin levels. These results demonstrate that insulin resistance is associated with elevated plasma leptin levels independent of body fat mass. However, plasma insulin itself does not acutely regulate leptin production.

Adult↗
PubMed1996

Radioimmunoassay of leptin in human plasma.

Recent studies suggest that leptin, the ob gene product absent in ob/ob mice, is a negative regulator of adiposity. We developed an RIA to measure human leptin in plasma or serum. The minimum detectable concentration by the assay is 0.5 microg/L leptin and the limit of linearity is 100 microg/L. Recovery of leptin added to serum was 99-104% over by the linear range of the assay. The RIA agreed reasonably well with rough quantification by Western blot (RIA = 0.90 blot + 3.7 microg/L, Sy/x = 10.9 microg/L). CVs within- and between-run ranged from 3.4% to 8.3% and from 3.6% to 6.2%, respectively. Variation in plasma leptin concentrations in specimens collected on consecutive mornings was large (CVs of 10.9% and 22.5%). After an overnight fast, leptin concentrations were similar to those 1-2 h after 1-2 meals. Plasma leptin concentrations in specimens from 83 lean and obese adults correlated directly with body mass index (BMI; kg/m2): r = 0.72, P <0.001. Correlations were significantly improved by separating results by gender (men r = 0.84, women r = 0.87; p <0.001). The increase in leptin concentrations with increasing BMI was greater in women than in men (slope 2.53 vs 0.97 microg/L per unit BMI, respectively). Leptin concentrations determined in lean subjects (BMI between 18 and 25) were higher in women (7.36 +/- 3.73 microg/L) than in men (3.84 +/- 1.79 microg/L) (P <0.001). Plasma leptin varied little with age and no significant difference was observed between whites and blacks. We conclude that: (a) plasma leptin concentrations are accurately and precisely measured by this new RIA; (b) leptin concentrations vary little due to short-term fasting, age, or race; but (c) plasma leptin concentrations are gender specific.

Adolescent↗
PubMed1995

A new plastic evacuated tube with plasma separator.

A new plastic-walled, evacuated blood collection tube (Terumo Venoject II) with plasma separator was evaluated for characteristics related to the use of plastic in place of glass. Plastic tubes were highly resistant to breakage through handling mishaps or failure during centrifugation. Higher centrifugation speed (13,600g) was well tolerated, and centrifugation times as short as 3 minutes at 13,600g effectively cleared plasma of cellular components, at high plasma yield. Plastic tubes were nearly completely combustible under incineration conditions commonly used for medical waste, and plastic tubes effectively retained vacuum during the typical shelf life of evacuated blood collection tubes. Collection of carbamezepine specimens in plastic tubes decreased levels an average of 6.8% compared to a heparinized glass tube control under conditions approximating routine use; levels of other drugs (phenytoin, phenobarbital, valproic acid, theophylline, and cyclosporine) were less significantly affected. This modest decrease appeared to result from a combination of an immediate interaction with the plastic surface of the tubes and a time-dependent interaction with the olefin-based separator. Modest but clear benefits, including reduction in specimen breakage, reduced centrifugation time and reduced solid waste after incineration derive from the use of plastic in place of glass in evacuated blood collection tubes.

Adolescent↗
PubMed1995

A new digoxin immunoassay substantially free of interference by digoxin immunoreactive factor.

We have evaluated the new Roche digoxin "On Line" procedure for use in a pediatric population with particular interest in the potential for interference by digoxin-like immunoreactive factor (DLIF). An initial study comparing digoxin values obtained with the new Roche procedure with determinations on an Abbott TDx, American Dade Stratus, and COBAS-FARA using Microgenics Cedia reagents, found good correlations with these established methods. The Roche method was suitably precise and utilized either serum or plasma. Interference by DLIF was assessed by analyzing specimens from patients not receiving digoxin but likely to contain DLIF, with the argument that non-zero values represent cross-reactivity of anti-digoxin antibodies with DLIF endogenous to these specimens. When specimens from neonates, women with second/third trimester pregnancies, and patients with renal and liver failure were assayed with the Roche, Stratus, and TDx methods, all three methods measured DLIF in some specimens, but the Roche method possessed the lowest overall DLIF interference. The modest extent of DLIF interference and the requirement of a small amount of specimen make the Roche method superior in monitoring digoxin in a pediatric population.

Digoxin↗
PubMed1995

Alkaline phosphatase: placental and tissue-nonspecific isoenzymes hydrolyze phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5'-phosphate. Substrate accumulation in carriers of hypophosphatasia corrects during pregnancy.

Hypophosphatasia features selective deficiency of activity of the tissue-nonspecific (liver/bone/kidney) alkaline phosphatase (ALP) isoenzyme (TNSALP); placental and intestinal ALP isoenzyme (PALP and IALP, respectively) activity is not reduced. Three phosphocompounds (phosphoethanolamine [PEA], inorganic pyrophosphate [PPi], and pyridoxal 5'-phosphate [PLP]) accumulate endogenously and appear, therefore, to be natural substrates for TNSALP. Carriers for hypophosphatasia may have decreased serum ALP activity and elevated substrate levels. To test whether human PALP and TNSALP are physiologically active toward the same substrates, we studied PEA, PPi, and PLP levels during and after pregnancy in three women who are carriers for hypophosphatasia. Hypophosphatasemia corrected during the third trimester because of PALP in maternal blood. Blood or urine concentrations of PEA, PPi, and PLP diminished substantially during that time. After childbirth, maternal circulating levels of PALP decreased, and PEA, PPi, and PLP levels abruptly increased. In serum, unremarkable concentrations of IALP and low levels of TNSALP did not change during the study period. We conclude that PALP, like TNSALP, is physiologically active toward PEA, PPi, and PLP in humans. We speculate from molecular/crystallographic information, indicating significant similarity of structure of the substrate-binding site of ALPs throughout nature, that all ALP isoenzymes recognize these same three phosphocompound substrates.

Alkaline Phosphatase↗
PubMed1995

Analytical artifacts in hematocrit measurements by whole-blood chemistry analyzers.

Compact analyzers suited to near-patient testing estimate hematocrit by measuring the conductivity of undiluted blood. We evaluated the accuracy of hematocrit determination of one such analyzer (Instrumentation Laboratory BGE analyzer) against an automated cell counter (EPC) and packed cell volume (PCV) microhematocrit. When specimens (n = 34) from outpatient and ward patients were analyzed with all three methods, the BGE analyzer correlated well with both EPC and PCV hematocrit determinations (BGE = 1.00 PCV + 0.3%, S(y)/x = 2.0%), suggesting that all three methods are similar in performance for most patients. However, a patient with increased plasma osmolality showed significant decreases in BGE and PCV hematocrits relative to the EPC method. The differences in hematocrit measurements could be reproduced by adding solutes to blood in vitro or by modifying the plasma osmolality of rats in vivo. Samples from patients undergoing cardiac surgery, whose blood had large changes in protein concentration, showed discrepancies between hematocrits by conductivity and other methods; similar effects could be produced by changes in protein concentration or in vitro addition of polyethylene glycol. We conclude that conductivity measurements provide accurate hematocrit results for physiologically normal subjects but not for some intensive-care and surgical patients.

Animals↗
PubMed1994

Glucose activates the multifunctional Ca2+/calmodulin-dependent protein kinase II in isolated rat pancreatic islets.

The influence of the insulin secretagogues, glucose and K+, to activate the multifunctional, Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in isolated rat pancreatic islets has been examined. Glucose (28 mM) and K+ (40 mM) were demonstrated to induce a 1.89 +/- 0.19- and 1.75 +/- 0.15-fold increase, respectively, in phosphorylation of a subunit of CaM kinase II immunoprecipitated by an anti-CaM kinase II alpha antibody. In intact islets, glucose and K+ also induced the generation of an autonomous, Ca2+/calmodulin-independent protein kinase II activity characteristic of autophosphorylated enzyme. Maximal activation, 2.9 +/- 0.2- and 3.0 +/- 0.5-fold for glucose and K+, respectively, relative to basal glucose control, was achieved at 2.5-5 min followed by a decline to near basal levels by 20 min. Glucose induced the production of autonomous CaM kinase II activity that, in terms of -fold stimulation, correlated closely with the extent of insulin release over a glucose concentration range of 3-28 mM. This stimulated activity was completely prevented by an inhibitor of glucose metabolism, mannoheptulose. These data demonstrate that the exposure of islets to stimulatory glucose concentrations activates CaM kinase II. The close correlation of enzyme activation with insulin secretion is consistent with the hypothesis that CaM kinase II plays an important role in the regulation of insulin secretion or related beta-cell processes.

Amino Acid Sequence↗
PubMed1994

Rapid measurement of serum pancreatic amylase.

A simple, rapid assay for the pancreatic isoenzyme of human serum amylase was developed. The assay utilized an immunoabsorbent prepared by coating latex beads with a monoclonal antibody specific for pancreatic amylase. Treatment of patient serum with immunoabsorbent removed pancreatic amylase, and measurement of residual amylase activity with standard total amylase methodology allowed estimation of the pancreatic amylase content. Extraction efficiency of pancreatic amylase was consistent at amylase concentrations up to 1,000 U/L (y = 0.97 x +16.7 U/L; r = 0.9995). The assay was standardized with purified pancreatic amylase added to neonatal serum (low endogenous activity). A comparison of patient specimen results with the results of a standard technique (cellulose acetate electrophoresis) yielded an excellent correlation (immunoabsorption result = 0.96 electrophoresis result + 1.2 U/L; r = 0.987). Salivary amylase did not interfere with the assay until levels exceeded 1,000 U/L. Daily analysis of a frozen serum pool yielded a coefficient of variation of 9.2% at mean pancreatic amylase value of 54 U/L (+/- 5 U/L). A normal range study found a strong influence of age, with pancreatic amylase levels increasing dramatically in the first 3 years of life, to stabilize at a range of 0-66 U/L thereafter.

Amylases↗
PubMed1994

Activation of calcium and calmodulin dependent protein kinase II during stimulation of insulin secretion.

Pancreatic islets contain an alloxan sensitive, calcium and calmodulin dependent protein kinase (CaM-PK) which may play an important part in the cellular control of insulin secretion. We have studied this activity in islets and the insulin secreting tumor cell line RINm5f with particular interest in the changes in kinase activity that accompany stimulation of secretion. Initial experiments showed that the CaM-PK activity enriched in microsomal preparations from RIN cells was similar to the islet cell kinase in terms of apparent endogenous substrates, Ca2+ and calmodulin dependence, and inactivation by alloxan. For studies of protein substrate specificity, tumor cell CaM-PK was isolated from other kinase activities and substantially purified by affinity chromatography with calmodulin-agarose. The major protein substrates of CaM-PK (54 kD and 57 kD) co-purified with the kinase activity, representing autophosphorylation of subunits of the enzyme. Exogenous substrates phosphorylated by these preparations included microtubule-associated protein 2, synapsin, and glycogen synthase; this pattern of substrate utilization identified the kinase as the Type II multifunctional kinase which has been extensively characterized in brain. A polyclonal antibody to rat brain CaM-PK II was employed to immunoprecipitate the kinase from RINm5f cells incubated with secretagogues to measure the effect of stimulation of secretion on autophosphorylation of CaM-PK (which reflects kinase activation). D-Glyceraldehyde (22 mM) and depolarizing concentrations of potassium increased autophosphorylation and insulin secretion in a parallel fashion. Potassium stimulated autophosphorylation was dose dependent and saturable, and was increased to near maximal levels at times as short as 1 min. These studies demonstrate that pancreatic islets and RINm5f cells contain a Type II CaM-PK which is activated during the secretion process.

Animals↗
PubMed1994

Detection of autoantibodies to amylase by ELISA: comparison of detection of macroamylase and free autoantibody.

New ELISAs for detecting macroamylase or free autoantibodies to amylase were tested with 48 samples that had been characterized by gel chromatography and electrophoresis. The macroamylase ELISA, with anti-IgG or anti-IgA for detection, detected macroamylase in 28 of 33 samples known to contain macroamylase (85% sensitivity), whereas the ELISA for free autoantibody to amylase was positive for only 11 samples. Specificities of both ELISAs were 93%. Among 28 true positives detected with the macroamylase ELISA, 22 contained IgA, 3 contained IgG, and 3 contained both immunoglobulins. Detection of IgM added no true positives. ELISA responses (y) were proportional to log [macroamylase concentration by chromatography (x)] from 0 to 1200 U/L: y = 5.15 x + 1.66; r = 0.72; Sy x = 1.65. As new tools for detecting macroenzymes consisting of enzyme-autoantibody complexes, the ELISAs show that some autoantibodies are detected more sensitively as antibody-antigen complexes than as free antibody.

Amylases↗
PubMed1994

Changes in plasma amino acid concentrations in response to HIV-1 infection.

Plasma concentrations of 21 amino acids were determined for 20 control subjects and 20 subjects infected with human immunodeficiency virus type 1 (HIV). Compared with the control subjects, the HIV-infected group had lower cystine, tryptophan, and methionine (decreased 67%, 52%, and 32%, respectively, P < 0.001 for each) and increased taurine (230%, P < 0.001) and lysine concentrations (30%, P < 0.001). Other amino acid concentrations changed modestly. Amounts of cystine, tryptophan, methionine, taurine, and lysine did not differ significantly between subgroups of HIV-infected subjects with > 200 (n = 6) or < 200 (n = 14) CD4+ lymphocytes per microliter, suggesting that the concentrations decrease soon after infection and change little thereafter. Activation of metabolism of cystine to taurine may explain reciprocal changes in these amino acids and known depletion of cystine and glutathione. The selective changes in amino acid profiles observed during HIV infection differ from those recognized for malnutrition or other pathological processes.

Acquired Immunodeficiency Syndrome↗