Glycaemic variability is associated with coronary artery calcium in men with Type 1 diabetes: the Coronary Artery Calcification in Type 1 Diabetes study.

AIMS: We investigated coronary artery calcium in association with glucose levels and variability measured using continuous glucose monitoring in adults with Type 1 diabetes in the Coronary Artery Calcification in Type 1 Diabetes study. METHODS: Coronary artery calcium was measured by electron beam tomography. The presence of any coronary artery calcium was analysed with respect to glucose levels [mean(T) (mean glucose), % of values < 3.9 mmol/l, > 10 mmol/l and either < 3.9 or > 10 mmol/l] and glycaemic variability [sd(T) (sd of all glucose values); sd(dm) (sd of the daily mean glucose levels) and sd(hh:mm) (glucose sd for a specified time of day, over all days)] using 3-5 days of continuous glucose monitoring from 75 subjects (45 women, 30 men), age 42 ± 9 years (mean ± sd) and diabetes duration of 29 ± 8 years using logistic regression. RESULTS: We observed significant associations between coronary artery calcium and mean(T) (OR = 4.4, 95% CI 1.1-18.6), % of values > 10 mmol/l (OR = 5.5, 95% CI 1.3-22.6), % of measures < 3.9 or > 10 mmol/l (OR = 5.7, 95% CI 1.3-24.9), sd(T) (OR = 4.7, 95% CI 1.1-19.7), sd(dm) (OR = 6.0, 95% CI 1.2-30.4) and sd(hh:mm) (OR = 4.0, 95% CI 1.1-15.4), among men, but none of these variables were associated with the presence of coronary artery calcium in women. CONCLUSIONS: We report the novel finding that subclinical atherosclerosis is associated with glucose levels and variability in men with Type 1 diabetes. The relationship of coronary artery calcium and glucose variability in Type 1 diabetes, and potential gender differences in this association, deserve further study.

Development of new methods to determine work disability in the United States.

The development of new methods to determine work disability for the United States Social Security Administration is described, including the fiscal and administrative background to the current and proposed methods. An introduction to the current disability determination process and description of its status is followed by a description of the original proposed plan for redesign of the process. In response to this plan, the authors participated in several research projects. An overview of some of the key research projects performed to improve the Social Security Administration disability determination process is provided.

Increased pulse wave velocity and shortened pulse wave transmission time in hypertension and aging.

We measured pulse wave velocity (PWV) and pulse transmission time (PTT) in 29 patients with hypertension, ranging in age from 37 to 73 years, in a series of 36 normal subjects with the same age range, and in an additional series of 44 normal subjects aged 18-35 years. PWV increases linearly with age for both normal subjects and patients with hypertension, with a corresponding significant decrease in PTT. There was a statistically highly significant (p < 0.001) increase in PWV in hypertension at all ages examined. The present simple noninvasive methods may be useful when evaluating risk factors for atherosclerosis and when evaluating response to therapeutic intervention.

Aldosterone stimulated differentiation of mouse 3T3-L1 cells into adipocytes.

We find that 1-10 nM aldosterone can induce differentiation of 3T3-L1 cells into adipose cells as evaluated by microscopic accumulation of fat droplets and quantitative measurement of triglycerides and of glycerol-3-phosphate dehydrogenase, an enzyme specific for adipocyte differentiation. Moreover, the aldosterone antagonist ZK91587 inhibits aldosterone-but not glucocorticoid-mediated differentiation of 3T3-L1 cells. Steroid binding assays with 3T3-L1 cells indicate the presence of specific binding sites for aldosterone. We conclude that there is an aldosterone receptor-mediated pathway for terminal differentiation of 3T3-L1 cells into adipose cells. Receptors for aldosterone have also been found in a variety of cells that do not function to regulate sodium and potassium transport. The aldosterone receptor may have a role in regulation expression of genes involved in differentiation of these cells.

Beta gamma subunits of guanine nucleotide-binding proteins and regulation of spontaneous receptor activity: thermodynamic model for the interaction between receptors and guanine nucleotide-binding protein subunits.

We used a thermodynamic model to examine the interactions between receptor, guanine nucleotide-binding protein (G protein), and their ligands. The model describes the interactions as multiple equilibria occurring between three distinct protein species (receptor, G alpha subunit, and G beta gamma complex) and two small ligands, i.e., agonist (which interacts with receptor) and guanine nucleotide (which binds to G alpha). The equilibrium distribution of free and complexed species is determined by the total concentration of the components, the affinities that govern the biomolecular reactions, and the allosteric interactions that ligands exert on each other when they are simultaneously bound to the same species. These allosteric factors are given in terms of free energy coupling. The model explains a number of experimental observations, as follows. (i) Both GTP and GDP can reduce agonist affinity, whereas the agonist enhances the net binding of GTP and diminishes that of GDP. (ii) G beta gamma is more effective in reducing agonist-independent than agonist-dependent receptor activity. (iii) Removal of guanine nucleotides increases the ratio between agonist-independent and -dependent activation of G protein. The model leads to a number of interesting predictions. (i) Not only G alpha but also G beta gamma has effects on hormone binding. (ii) As long as the distribution of protein species is [G beta gamma] > [G alpha] > [receptor] (as often observed in the cell membrane), small changes in the concentration of G beta gamma do not alter the overall response induced by agonist. (iii) Agonist activity examined at low concentrations of guanine nucleotide is inevitably different from that observed at high concentrations, typical of intact systems. (iv) Differences in potencies and maximal effects for various guanine nucleotide analogues may reflect differences in their coupling constants that are experimentally measurable. The present model suggests several experimentally testable hypotheses that could be important in elucidating the activation mechanism and regulatory flexibility of G protein-dependent transduction systems.

A model for the effect of estrogen antagonists on cooperative estradiol binding.

Partial agonists such as estriol and estrone have been reported to diminish or even eliminate the upward convexity of the Scatchard plot of the binding of labeled estradiol to estrogen receptor. This has been interpreted as agonist interference with the receptor dimerization induced by estradiol. In order to investigate how a partial agonist or antagonist might interfere with dimerization we have developed a theoretical mass-action law model, where soluble receptors can dimerize and bind to two different ligands. Special attention was devoted to manifestations of positive cooperativity to determine whether they could be modified by competition with a second ligand. This was done using a computer program that evaluated a large set of combinations of affinity constants in an effort to explore all possible situations. The model could reproduce the effect of a second ligand on the cooperative binding of estradiol to the estrogen receptor but only if the second ligand was anticooperative, which is not the case of estriol, estrone and tamoxifen. Furthermore, even when the Scatchard plot was linear, the model still required dimerization of the receptor in most of the cases, showing that the addition of an antagonist may eliminate the upward curvature of the Scatchard without truly eliminating dimerization or cooperativity. We conclude that the effect of a second ligand on the binding of labeled estradiol to estrogen receptor is not necessarily due to interference with dimerization and/or cooperativity. The inability of this model to fully explain the published data for estriol, estrone, clomiphene, and tamoxifen suggests that a more complex mechanism is involved.

Mouse mammary epithelium produces a soluble heat-sensitive macromolecule that inhibits differentiation of 3T3-L1 preadipocytes.

Coculture of normal mouse mammary gland (NMMG) epithelial cells with 3T3-L1 preadipocytes resulted in inhibition of triglyceride accumulation. This inhibition was also observed when the NMMG cells were grown in inserts and placed within a 100-mm dish containing confluent 3T3-L1 cells. As the number of NMMG-containing inserts was increased, there was a progressive decline in triglyceride content of the 3T3-L1 cells. Conditioned medium from NMMG cells also resulted in a dose-dependent inhibition of adipocyte formation, and when concentrated 10-fold by passage through a filter with a cutoff of 30 kDa, all of the inhibitory activity was recovered. Heating the concentrated conditioned medium at 98 degrees C for 30 min resulted in complete loss of activity. Of several peptides tested, transforming growth factor-beta, platelet-derived growth factor, tumor necrosis factor, interleukin 6, and basic fibroblast growth factor showed inhibitory activity, whereas epidermal growth factor, insulin-like growth factor I, and transforming growth factor-alpha did not.

Progestins stimulate the differentiation of 3T3-L1 preadipocytes.

The effect of progesterone on the differentiation of the 3T3-L1 preadipocytes was investigated and compared with other sex steroids (estradiol and testosterone), with cortisol, with the synthetic progestin R5020 and with the progestin/glucocorticoid antagonist RU38486. At 10(-8) M, progesterone stimulated the activity of glycerol-3-phosphate dehydrogenase and triglyceride deposition. Progesterone, R5020, cortisol, and RU38486 increased triglycerides about 2-fold at 10(-7) M. Only minimal effects were observed with testosterone and estradiol even at 10(-6) M. When the cells were cultured in presence of 10(-5) M metyrapone the effect of progesterone was unchanged, suggesting that the progesterone was not metabolized to a glucocorticoid. Progesterone, R5020 and RU38486 competed efficiently with [3H]dexamethasone for the glucocorticoid receptor in 3T3-L1 cytosol. These results indicate a significant, reproducible dose-dependent effect of progestins on differentiation of the preadipocytes, which appears to be mediated via the glucocorticoid receptor.

Drug efficacy at guanine nucleotide-binding regulatory protein-linked receptors: thermodynamic interpretation of negative antagonism and of receptor activity in the absence of ligand.

The mutual effects that a hormonal ligand (H) and a guanine nucleotide regulatory protein (G protein) exert on each other when simultaneously occupying distinct sites of the receptor molecule (R) can be viewed as the molecular mechanism of drug efficacy. These effects are predictable on the basis of a model assuming that the ternary complex between the three partners (HRG) reaches equilibrium in the membrane [J. Biol. Chem. 255:7108-7117 (1980)]. Ligands can be classified as agonists, neutral antagonists, or negative antagonists, depending on whether they enhance, leave unchanged, or reduce, respectively, the spontaneous tendency of R to interact with G. Using this model and the assumption that the G protein response observed in membranes reflects the sum of ligand-independent (RG) and ligand-dependent (HRG) receptor-G protein complexes, we can explain virtually all the phenomenology reported earlier for opioid receptor-mediated stimulation of GTPase, i.e., 1) existence of ligands with both "positive" and "negative" intrinsic activity (the latter termed negative antagonists), 2) equipotency of neutral antagonists for the competitive blockade of the responses elicited both by agonists and by negative antagonists, and 3) apparent heterogeneity of binding sites for the binding isotherms of negative antagonists. The ternary complex model can also explain the differential effects of sodium on ligand binding and ligand-dependent GTPase activity, if we assume that this ion reduces the stability constant between receptor and G protein in membranes. Computer simulations predict that a negative antagonist exhibits a discrepancy between "biological" Ki (obtained by Schild plots) and true dissociation constant for the receptor, which increases as the fraction of "precoupled" receptors in the membrane increases. The demonstration of negative antagonism is definitive evidence for the existence of receptor coupling (hence activity) in the absence of ligand. Using this experimental paradigm, we show here that spontaneous receptor activity occurs in isolated membranes but not in intact NG108-15 cells.

Quantitative characterization of multiple binding sites for phencyclidine and N-allylnormetazocine in membranes from rat and guinea pig brain.

Quantitative ligand binding studies have been used to characterize binding sites for N-allylnormetazocine ((+)SKF10,047) (SKF), 1-(1-phenylcyclohexyl) piperidine (PCP), N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) and haloperidol in membranes from the brain of rat and guinea pig under conditions which permitted simultaneous analysis of the binding of both PCP and SKF. Using four labelled ligands (SKF, TCP, PCP and haloperidol), each displaced by the corresponding four unlabelled ligands, four classes of binding sites were observed in membranes from the brain of the rat, corresponding to sigma (sigma), two classes of PCP sites (PCP1, PCP2) and dopamine (D2) sites. The sigma site was suppressed by 50 nM haloperidol, while the PCP1 and PCP2 sites were not. These results were confirmed by studies employing a self- and cross-displacement design and dose-response surfaces for SKF and TCP, with and without blockade by haloperidol of the sigma site. Using mathematical modelling, employing the program LIGAND, it was possible to reject simpler models involving a common "PCP/sigma" site or a model involving only two classes of sites (sigma and PCP). Similar methods were used to identify two classes of sigma binding sites and two classes of PCP binding sites, in membranes prepared from the brain of the guinea pig. The relative potencies of 18 ligands for displacement of (+)[3H]SKF10,047 and [3H]TCP were compared: there were significant qualitative and quantitative differences in the "sigma" binding sites in the brain of rat and guinea pig, while the PCP binding sites were very similar in the two species.

Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans.

The proteoglycans (cell-associated and culture media) in 3T3-L1 preadipocytes in culture were analyzed before and during differentiation into adipocytes. Cells were metabolically labeled with [35S]sulfate and [3H] glucosamine for 24 h and then extracted and analyzed. There was a 1.68 +/- 0.07-fold increase in the 35S in medium proteoglycan during differentiation, whereas cell-associated proteoglycan radioactivity showed no increase. Analyses of radiolabeled molecules using ion-exchange chromatography, gel filtration, and high performance liquid chromatography after enzymatic or alkaline digestion indicated that all of the 35S label was recovered as two major species of chondroitin 4-sulfate proteoglycans (CSPG-I and CSPG-II) and 7% as heparan sulfate proteoglycan. CSPG-I has a mass of approximately 970 kDa with multiple chondroitin sulfate chains (average of 50 kDa each) and a core protein of approximately 370 kDa including oligosaccharides. CSPG-II has a mass of 140 kDa with one or two chondroitin sulfate chains (average of 68 kDa each) and a core protein of 41 kDa including oligosaccharides. CSPG-I appears to be similar to versican, whereas CSPG-II is similar to decorin and/or biglycan, found in other fibroblastic cells. Cell differentiation was associated with a specific increase in CSPG-I (4.0 +/- 0.2-fold in media and 3.2 +/- 0.5-fold in the cell-associated form). This system should facilitate study of the functional roles of proteoglycans during growth and differentiation.

A pharmacodynamic approach to optimizing insulin therapy.

We have developed a program for simulation and optimization of insulin therapy in patients with insulin-dependent diabetes. The program, denoted GLUCOJECT, is based on a physiologic model of minimal complexity, which describes the pharmacokinetics of absorption and clearance of subcutaneous insulin and the dynamics of glucose utilization as dependent on both prevailing glucose and insulin levels. With self-monitored glucose values and insulin doses collected with one of several commercially available memory meters, GLUCOJECT reconstructs an average or 'typical' daily plasma glucose and insulin profile and displays them in a graph. The program then calculates the expected rate of glucose utilization, which permits calculation of the rate of glucose entry into plasma from both endogenous (hepatic) and exogenous (dietary) sources. In turn, this allows one to calculate an 'ideal' plasma insulin profile required to maintain a relatively constant 'ideal' plasma glucose level. GLUCOJECT can evaluate several different insulin regimens involving various combinations of short-, intermediate- and long-acting insulins, and select the one(s) most closely approximating the ideal or optimal insulin profile, using a least-squares criterion. For any optimized insulin regimen, GLUCOJECT calculates and displays the predicted time course of plasma glucose. These features make the program attractive as an educational tool for both patients and health care professionals and could potentially assist in the management of patients with insulin-dependent diabetes.

Use of the receiver operating characteristic curve to evaluate sensitivity, specificity, and accuracy of methods for detection of peaks in hormone time series.

We utilize the "Receiver Operating Characteristic" to describe the relationship between sensitivity and specificity as the threshold for peak detection is varied systematically, to provide objective comparison of the performance of methods for detection of episodic hormonal secretion. A computer program was used to generate synthetic data with peaks with variable durations, with constant or variable height, shape and/or interpulse interval. This approach was used to compare the CLUSTER and DETECT programs. For both programs, the observed false positive rates estimated using signal-free data were in good agreement with the nominal rates, but in the presence of signal the observed false positive rates were systematically lower. Sensitivity increases with increasing signal/noise ratio, as expected. Program DETECT, using its standard options, provided excellent sensitivity (90-100%) with very low false positive rate under all conditions tested. Its performance could be further improved by the use of a more stringent definition of a peak requiring the presence of "UP" followed by a "DOWN". The CLUSTER program was found to have very poor sensitivity when using the "local variance" option. Use of the true fixed standard deviation or percent coefficient of variation resulted in a modest improvement. Optimal performance of program CLUSTER was obtained by the use of the best of 3 variance models, testing 12 different cluster sizes (from 1x1 to 4x4) and selecting the best among these: under these conditions it can achieve high sensitivity (90-100%) for very low observed false positive rate, such that its performance was comparable to that of DETECT. The methods developed and illustrated here should permit the definitive characterization and validation of the performance of any one method, the objective comparison of the relative performance of two or more methods for analysis of pulsatile hormone levels for episodic hormone secretion, and lead to the improvement of algorithms for peak detection.

Objective assessment of concordance of secretory events in two endocrine time series.

A new objective method is presented for investigating the presence of a temporal relationship between episodic release of two hormones. The two time series of hormone concentrations are first analysed by an objective method for peak detection. Both data series are then transformed into "quantized" or discretized series by recording the occurrence of a hormone pulse as an "event", characterized by the onset, the maximum, or another unique feature. The two quantized series are then matched, and the number of concordant events and discordant events are counted. Each point in series A is compared with a "time-window" of a selected number of points in series B, to accommodate small degree of mismatch between events in the two series. An index of concordance is computed, compensating for any spurious random coincidence: the "Specific Concordance", to evaluate the frequency of concordant events in excess of those expected on the basis of chance alone. This calculation is systematically repeated, interposing a range of time-lags between the two series. A graph of Specific Concordance versus time-lag indicates the time-lag corresponding to a maximal concordance. Simulations of random series of events are performed, and their degree of concordance is evaluated in a similar fashion, thus generating frequency distributions of Specific Concordance values under the null hypothesis of no temporal relationship. This permits the selection of criteria for statistical significance at any desired p-level, for one or many lag times, and for one or multiple subjects. Various degrees of concurdance can also be stimulated to evaluate the performance (sensitivity, statistical power) of this approach. These methods have been implemented as a collection of short microcomputer programmes, and applied to the study of the temporal relationship between beta-endorphin and cortisol in normal subjects sampled every 10 min for 24 h. This analysis demonstrated concordance between events in the two series, with synchronous occurrence of beta-endorphin and cortisol release events significantly more frequently than expected on the basis of random association (p less than 0.01).

Timing of the carotid arterial sounds in normal adult men: measurement of left ventricular ejection, pre-ejection period and pulse transmission time.

The first and second carotid arterial sounds (CaS1 and CaS2) were recorded simultaneously with the aortic valve echocardiogram, carotid pulse wave contour, heart sounds, and electrocardiogram in 27 healthy, normal subjects. The mean intervals between the onset of the QRS complex and the onsets of the first and second major components of the carotid arterial sounds were Q-CaS1 = 131 +/- 15 ms and Q-CaS2 = 412 +/- 36 ms, respectively. The mean delay of CaS1 after aortic valve opening was 43 +/- 6 ms, while the delay of CaS2 after aortic valve closure was 43 +/- 7 ms. The onset of CaS1 and CaS2 are exactly coincident with the upstroke and with the dicrotic notch of the carotid pulse wave contour. The recording of the carotid arterial sounds, heart sounds, and ECG has enabled us to measure systolic time intervals including the pre-ejection period, left ventricular ejection time, and pulse transmission time more easily than using the conventional method involving the carotid arterial pulse wave contour. The new approach provides accuracy and precision comparable to that of the previous methods.

Improved diabetic prognosis following telecommunication and graphical processing of diabetic data.

The thesis that an integrated telecommunications/reporting system would affect diabetic prognosis was tested. Over fifteen months a double crossover study compared traditional diaries versus graphical display of telecommunicated blood glucose data. Significant drops in glycohemoglobin were observed in both groups during the telecommunications period, while no significant drops were observed in the groups while diaries were employed.

Pulsatile secretion of luteinizing hormone in agonadal men before and during testosterone replacement therapy.

UNLABELLED: We have reevaluated the question regarding the pulsatile pattern of LH secretion in agonadal men before and following testosterone replacement therapy. Five normal males were used as a reference group and four agonadal men were studied before and during replacement therapy with testosterone enanthate. All the subjects were sampled every 5 min for 12 h (08:00 to 20:00). Data were analyzed using the statistically based and validated pulse detection program DETECT. The normal subjects showed an LH pulse frequency of 10.2 +/- 1.7 peaks/12 h (mean +/- SEM) and a mean duration of 48.8 +/- 14 min, while in agonadal patients without testosterone replacement the frequency of LH peaks (27.5 +/- 2 peaks/12h) was significantly higher than for normal subjects (p less than 0.05), and the mean duration of peaks was lower than in controls (17.2 +/- 1.2 min; p less than 0.01). Following chronic testosterone enanthate replacement therapy (200 mg im every two weeks) these patients showed an increase in the duration and a significant reduction in the frequency of LH peaks (from 27.5 +/- 2 to 18.2 +/- 2.1 peaks/12 h; p less than 0.01) but pulse frequency remained significantly higher than for normal subjects (p less than 0.01). This finding is independent of the choice of p values for false positive detection rate (p = 0.01 or p = 0.005), but it does depend on sampling frequency and is influenced by large (four-fold) changes in the thresholds for peak detection. Using a "discrete deconvolution" technique we estimated the instantaneous secretory rate (ISR) for the two groups of patients. The results using ISR corroborated the findings obtained using analysis of observed plasma LH measurements. ISR computation also showed that the duration of the secretory events of the gonadotropes is significantly shorter (p less than 0.01) than the one estimated on plasma concentration, both in normal subjects and in agonadal patients before and during testosterone administration. IN CONCLUSION: LH pulse frequency observed in basal conditions in agonadal men was much higher than previously reported in primary testicular failure; during conventional testosterone replacement therapy LH pulse frequency of agonadal men was significantly reduced but still higher (p less than 0.01) than in normal men. This finding is probably related to the subnormal plasma levels of testosterone found in agonadal men during the replacement therapy; the analysis of data using a sampling interval of 10 min gave results similar to previous reports, confirming that the choice of sampling interval can markedly affect the evaluation of frequent LH pulsatile secretion.

Computerized estimation of size of nucleic acid fragments using the four-parameter logistic model.

We have developed improved methods for statistical estimation of the size of linear duplex DNA after continuous- or pulsed-field electrophoresis in agarose and polyacrylamide gels. We employ the four-parameter logistic model to describe the smooth, symmetrical, sigmoidal relationship between electrophoretic mobility (distance migrated) and log of molecular size (kb): distance = a - d/1 + (size/c)b + d. The four parameters (a,b,c,d) are estimated by nonlinear least-squares curve fitting using the Marquardt-Levenberg algorithm, where a represents an upper plateau, b a slope factor, c the midpoint, and d the lower plateau. Estimates of size for unknown species are accompanied by estimates of the standard error (SE) or coefficient of variation (%CV). A plot of SE and %CV versus size results in a "precision profile" which objectively defines the useful range of the calibration curve. This logistic relationship is more general than the rectangular hyperbola or linear methods, provides excellent goodness of fit, and can be used as a "global" method for the entire calibration curve, rather than as a "local" method for small segments of the curve.(ABSTRACT TRUNCATED AT 250 WORDS)