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Results for “cell deconvolution”

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PubMed2003

Analysis of subcellular calcium signals in T-lymphocytes.

Subcellular Ca(2+) signals were analysed in Jurkat and peripheral human T-lymphocytes by confocal Ca(2+) imaging employing an off-line deconvolution method. Stimulation of the TCR/CD3 complex in T-lymphocytes resulted in a series of subcellular pacemaker Ca(2+) signals preceding the first global Ca(2+) signal. The pacemaker signals occurred in a cytosolic "trigger" zone, which is localised close to the plasma membrane. The pacemaker signals were almost independent of extracellular Ca(2+) as shown by measurements in the absence of extracellular Ca(2+), or in the presence of the Ca(2+) channel blocker SK-F 96365. Analysis of the confocal Ca(2+) images revealed characteristic amplitudes of 82 +/- 30 to 109 +/- 21 nM, signal diameters between 2.5 +/- 0.9 and 3.5 +/- 1.5 microm and frequencies between 0.235 and 0.677 s(-1). Taken together, our data constitute the first analysis of subcellular Ca(2+) signals in T cells and indicate that the pacemaker Ca(2+) release events, which are necessary for the development of the global Ca(2+) signal, are composed of Ca(2+) release both from inositol 1,4,5-trisphosphate- and ryanodine receptors.

Calcium↗

PubMed2006

Mechanisms of recovery from type 2 diabetes after malabsorptive bariatric surgery.

Currently, there are no data in the literature regarding the pathophysiological mechanisms involved in the rapid resolution of type 2 diabetes after bariatric surgery, which was reported as an additional benefit of the surgical treatment for morbid obesity. With this question in mind, insulin sensitivity, using euglycemic-hyperinsulinemic clamp, and insulin secretion, by the C-peptide deconvolution method after an oral glucose load, together with the circulating levels of intestinal incretins and adipocytokines, have been studied in 10 diabetic morbidly obese subjects before and shortly after biliopancreatic diversion (BPD) to avoid the weight loss interference. Diabetes disappeared 1 week after BPD, while insulin sensitivity (32.96 +/- 4.3 to 65.73 +/- 3.22 mumol . kg fat-free mass(-1) . min(-1) at 1 week and to 64.73 +/- 3.42 mumol . kg fat-free mass(-1) . min(-1) at 4 weeks; P < 0.0001) was fully normalized. Fasting insulin secretion rate (148.16 +/- 20.07 to 70.0.2 +/- 8.14 and 83.24 +/- 8.28 pmol/min per m(2); P < 0.01) and total insulin output (43.76 +/- 4.07 to 25.48 +/- 1.69 and 30.50 +/- 4.71 nmol/m(2); P < 0.05) dramatically decreased, while a significant improvement in beta-cell glucose sensitivity was observed. Both fasting and glucose-stimulated gastrointestinal polypeptide (13.40 +/- 1.99 to 6.58 +/- 1.72 pmol/l at 1 week and 5.83 +/- 0.80 pmol/l at 4 weeks) significantly (P < 0.001) decreased, while glucagon-like peptide 1 significantly increased (1.75 +/- 0.16 to 3.42 +/- 0.41 pmol/l at 1 week and 3.62 +/- 0.21 pmol/l at 4 weeks; P < 0.001). BPD determines a prompt reversibility of type 2 diabetes by normalizing peripheral insulin sensitivity and enhancing beta-cell sensitivity to glucose, these changes occurring very early after the operation. This operation may affect the enteroinsular axis function by diverting nutrients away from the proximal gastrointestinal tract and by delivering incompletely digested nutrients to the ileum.

Adiponectin↗

PubMed2006

Spatial resolution in infrared microspectroscopic imaging of tissues.

Spatial resolution is one of the most critical measurement parameters in infrared microspectroscopy. Due to the distinct levels of morphologic heterogeneity in cells and tissues the spatial resolution in a given IR imaging setup strongly affects the character of the infrared spectral patterns obtained from the biomedical samples. This is particularly important when spectral data bases of reference microspectra from defined tissue structures are collected. In this paper we have also pointed out that the concept of spatial resolution in IR imaging is inseparable from the contrast. Based on infrared microspectroscopic transmittance data acquired from an USAF 1951 resolution target we have demonstrated how the spatial resolution can be determined experimentally and some numbers for the spatial resolution of popular IR imaging systems are provided. Finally, we have presented a new computational procedure which is suitable to improve the spatial resolution in IR imaging. A theoretical model of 3D-Fourier self-deconvolution (FSD) is given and advantages or pitfalls of this method are discussed. Based on synchrotron IR microspectroscopic data we have furthermore demonstrated that the technique of 3D-FSD can be successfully applied to increase the spatial resolution in a real IR imaging setup.

Animals↗

PubMed1999

A preponderance of basic luteinizing hormone (LH) isoforms accompanies inappropriate hypersecretion of both basal and pulsatile LH in adolescents with polycystic ovarian syndrome.

We recently demonstrated that adolescent girls with polycystic ovarian syndrome (PCOS) exhibit augmented LH secretion due to an increase in immunofluorometric and deconvolution-estimated LH secretory burst mass and pulse frequency. Concurrently, we inferred either a prolongation of apparent (endogenous) LH half-life or elevated basal (nonpulsatile) LH release in PCOS. The in vivo half-life of LH molecules can be affected by the oligosaccharide side-chains, which also modify in vitro bioactivity and electrostatic change. Accordingly, as a surrogate estimator of altered endogenous LH half-life and/or biopotency in PCOS, we characterized the isoelectric properties of secreted LH isoforms and determined their in vitro biological activity in adolescent girls with PCOS compared with healthy age-matched eumenorrheic controls. To this end, 12-h (overnight) serum samples from PCOS patients (n = 12) and normal adolescents (n = 10) were pooled by subject. Bioactive LH concentrations were then quantitated in a rat Leydig cell in vitro bioassay, and immunological activity was determined by immunofluorometry. The distribution of LH isoforms was evaluated by preparative chromatofocusing (pH window, 10.5 to <4.0) of samples further combined to yield three independent serum pools for each of the patient and control groups. Fasting serum concentrations of 17-hydroxyprogesterone (17-OHP), androstenedione, testosterone, estrone, estradiol, and sex hormone-binding globulin were determined as possible endocrine correlates of LH isotypes. Mean serum concentrations of immunoreactive and bioactive LH in adolescents with PCOS were 3 and 2 times higher than values in controls: immunoreactive: PCOS, 7.8+/-0.9; controls: 2.6+/-0.3 IU/L (P < 0.001); and bioactive: PCOS, 52+/-10; controls, 25+/-4.1 IU/L (P = 0.002), respectively. Bioactive LH concentrations correlated positively with 17-OHP (P = 0.022), androstenedione (P = 0.012), and testosterone (P = 0.046) concentrations in PCOS. Chromatofocusing of LH isoforms disclosed greater LH immunoreactivity at pI values greater than 8 and 7.99-7.0 in adolescents with PCOS compared with controls (P = 0.031). The percentage of basic LH isoforms was related positively to serum concentrations of 17-OHP (P = 0.032), androstenedione (P = 0.046), and testosterone (P = 0.040). In conclusion, the present isotype analysis demonstrates elevated in vitro LH bioactivity and a preponderance of basic LH isoforms in girls with PCOS. Since previously reported heterologous in vivo assays of LH kinetics point toward accelerated removal of such alkaline isotypes, our findings would favor the earlier alternative hypothesis of inappropriate hypersecretion of basal (interpulse) LH rather than prolongation of the LH half-life as the mechanism for elevated interpulse serum LH concentrations in adolescents with PCOS. In ensemble, the foregoing data thus suggest 3-fold amplification of basal LH secretion as well as both a heightened amplitude and frequency of the pulsatile mode of LH release in PCOS.

17-alpha-Hydroxyprogesterone↗

PubMed1979

X-ray microanalysis of pyroantimonate-precipitable cations.

Modifications of the Komnick potassium (pyro)antimonate precipitation method have been widely used for the subcellular localization of a variety of cations. The identity of cations precipitated with this method has often been controversial, and it is therefore important to establish definitive criteria for identifying precipitated cations in situ. In the present study, we have precipitated antimonate salts in vitro and examined the salts both in crystalline powder form and after embedment in Epon, using energy-dispersive X-ray microanalysis, in an attempt to identify biologically important antimonate-precipitable cations. We have found that the cations sodium, magnesium, and calcium, if present in physiological concentrations, will precipitate antimonate under "standard" conditions (2.5% antimonate, pH 7.2 - 7.4, in the presence of 1% OsO4). Characteristic X-ray emissions were observed for sodium and magnesium, as well as for lead similarly precipitated, but Lalpha and Lbeta emission peaks from antimony interfered with calcium identification and necessitated complex computerized deconvolution or peak stripping to determine the presence of a calcium peak. Precipitates of sodium did not contain appreciable potassium, whereas variable amounts of potassium were present in precipitates of calcium and lead, depending upon the extent of washing prior to dehydration. Sizeable potassium peaks were consistently present in even well washed magnesium precipitates. X-ray spectra of standardized precipitates were found useful as an aid in interpreting the more complicated spectra obtained from tissue samples.

Animals↗

PubMed1999

Mechanisms subserving the physiological nocturnal relative hypoprolactinemia of healthy older men: dual decline in prolactin secretory burst mass and basal release with preservation of pulse duration, frequency, and interpulse interval--a General Clinical Research Center study.

Increasing age is accompanied by decrements in randomly obtained, fasting, or frequently sampled serum PRL concentrations. The precise neuroendocrine mechanisms underlying such relative hypoprolactinemia in aging are incompletely understood. In the present study, we sampled blood at 2.5-min intervals overnight in 11 young (aged 21-34 yr) and 8 older (aged 62-72 yr) healthy men for subsequent chemiluminescence-based assay of serum PRL concentrations. The mean (+/- SEM) serum PRL concentration was significantly reduced at 4.3 +/- 0.78 microg/L in older men compared with 9.5 +/- 1.2 microg/L in young volunteers (P = 0.0049). PRL concentrations correlated with serum testosterone (r = 0.473; P = 0.041), dehydroepiandrosteroen sulfate (r = +0.455, P = 0.05), and insulin-like growth factor I (r = 0.494; P = 0.032) levels. Deconvolution analysis was used to evaluate combined pulsatile and basal modes of PRL secretion. In older men, discrete PRL secretory bursts were marked by a significantly (2.4-fold) attenuated mass of hormone secreted per burst (amount of PRL secreted per unit distribution volume), viz. 1.6 +/- 0.23 (older) vs. 3.9 +/- 0.57 microg/L (young; P < 0.01). In contrast, PRL secretory burst frequency, interpulse interval, and pulse duration were invariant of age. Concomitantly, basal PRL secretion was reduced by 2-fold in older subjects, namely to 0.00030 +/- 0.00027 (older) vs. 0.00065 +/- 0.0002 microg/L/min (young; P < 0.01). The amount of total PRL secretion that was pulsatile averaged 82 +/- 5.3% in young and 99 +/- 0.13% in older men (P = 0.012), indicating preferential loss of the basal mode of PRL release in aging. Assuming that basal PRL secretion mirrors functional pituitary lactotroph cell secretory mass, whereas pulsatile PRL release reflects effective (net) intermittent hypothalamic drive to responsive lactotroph cells, then our results suggest both an attrition in lactotroph cell mass and an impoverishment of net positive hypothalamic (agonistic) input to lactotrophs in older men. Given the multiple roles of PRL reported in experimental animals (e.g. on the one hand to support immune function and adrenal androgen biosynthesis and on the other hand to activate intraprostatic growth factors), we suggest that the nocturnal relative hypoprolactinemia observed in healthy aging men may have both adaptive and maladaptive clinical implications to target tissues.

Adult↗

PubMedDate not supplied

Exploring the mechanisms of vascular smooth muscle tone with highly specific, membrane-permeable inhibitors of cyclic GMP-dependent protein kinase Ialpha.

The structural similarity of cyclic GMP-dependent protein kinase (cGPK) and cyclic AMP-dependent protein kinase (cAPK) has made it difficult to study cGPK pathways independent of those mediated by cAPK, primarily due to the lack of potent and selective cGPK inhibitors. We recently reported a novel peptide library screen specifically designed to select for tight-binding peptides that identified selective inhibitors of cGPK [Proc Natl Acad Sci USA, 97 (2000) 14772]. Iterative deconvolution of octameric library arrays on paper identified the sequence LRK(5)H (W45). Binding of W45 to cGPK resulted in selective inhibition of the kinase, with K(i) values of 0.8 microM and 560 microM for cGPK and cAPK, respectively. Cellular internalization of highly charged W45 was accomplished by N-terminal fusion of membrane translocation sequences from either the human immunodeficiency virus tyrosine aminotransferase protein (47-59) DT-2 or from the Drosophila Antennapedia homeodomain (43-58) DT-3, respectively. For both fusion peptides, DT-2 and DT-3, we observed a potentiating effect with respect to the inhibitory potency, with K(i) values 40- to 80-fold lower than W45. Fluorescein-labeled DT-2 and DT-3 demonstrated rapid translocation through the cytosol and nuclei in a time-dependent manner using cultured cells and intact tissue samples (cerebral arteries). The physiological effects of DT-2 and DT-3 as selective cGPK inhibitors in smooth muscle were studied in small intact arteries. Nitric oxide, a cyclic GMP/cGPK activator, elicited a concentration-dependent dilation of isolated rat cerebral arteries, which was markedly inhibited by DT-2 and DT-3. Collectively, these results indicate that DT-2 and DT-3 effectively inhibit nitric oxide-induced vasodilation, further emphasizing the central role for cGPK in the modulation of vascular contractility.

Animals↗

PubMed1990

A statistical test for demonstrating a presynaptic site of action for a modulator of synaptic amplitude.

A statistical technique for demonstrating a presynaptic site of action for a modulator of synaptic amplitude was developed and tested. It requires that multiple measurements of peak synaptic amplitude be made under control and test conditions. The ratio of the coefficients of variation (CV) obtained under test and control conditions is calculated. A method was developed for determining the confidence interval for the CV ratio (CVR) statistic based on the null hypothesis that the synaptic modulation is purely postsynaptic. If the measured CVR falls outside the confidence interval, this implies that the modulator of synaptic amplitude is, at least in part, acting at a presynaptic site. The effectiveness of the technique and its limitations were investigated using Monte Carlo simulations. It was found to be sensitive and reliable under a variety of realistic recording conditions. The test was effective even in the presence of simulated presynaptic rundown of the synaptic response. Conventional deconvolution analysis was also applied to the Monte Carlo simulations and was found to be an inadequate indicator of the site of synaptic modulation when the discrete amplitude components were not well resolved. The CVR technique was applied to excitatory postsynaptic currents (epsc) recorded between pairs of cultured hippocampal neurones in control and test media containing 1 mM Ca2+ and 2 mM Ca2+, respectively. Test conditions increased the average synaptic amplitude, and the statistical analysis indicated that this modulation was produced by an action at a presynaptic site.

Animals↗

PubMed2000

Two comparisons of the performance of positional scanning and deletion synthesis for the identification of active constituents in mixture combinatorial libraries.

Two libraries of 120 compounds each were prepared as individual compounds and as full mixtures. The corresponding scanning and deletion synthesis deconvolution libraries were prepared and tested (L-1210, IC(50)) alongside the individual compounds and mixture libraries. This testing, where the properties of each compound in the mixtures were known, was used to compare the performance of scanning and deletion deconvolution libraries. Each has its own intrinsic strengths, with the former being capable of identifying multiple hits at the expense of accurately identifying the most potent library member, while the latter typically is more sensitive to identifying the most potent hit but at the expense of differentiating weaker activities. The protocols complement one another and together more thoroughly identify potent library members.

Animals↗

PubMed1998

Leukocyte activation does not mediate myocardial leukocyte retention during endotoxemia in rabbits.

Our goal was to determine whether coronary leukocyte retention after endotoxin infusion was due primarily to leukocyte activation. Leukocytes were activated by infusion of endotoxin into 12 blood donor rabbits. Separately, 12 isolated rabbit hearts were perfused with blood from an endotoxemic support rabbit to expose coronary endothelium to an inflammatory stimulus. During an infusion of 20 ml of donor blood into the isolated heart, the coronary transit time of leukocytes was determined by deconvolution of multiple measurements of injectate and collected leukocyte concentrations. With no leukocyte activation or inflammatory stimulation of endothelium, leukocyte transit time was 9.2 +/- 3.5 s, and 11.6 +/- 4.1 x 10(6) leukocytes were retained in the coronary circulation. Leukocyte activation alone did not alter transit time (9.8 +/- 3.2 s) or retention (9.3 +/- 4.6 x 10(6) leukocytes). Inflammatory stimulation of endothelium with and without leukocyte activation increased transit time (18.0 +/- 3.6 and 18.9 +/- 3.8 s, respectively; P < 0. 05) and retention (24.8 +/- 8.4 and 25.3 +/- 6.8 x 10(6) leukocytes, respectively; P < 0.05) to the same extent. Differential counts showed that neutrophils (but not lymphocytes) were slowed and retained. Inflammatory stimulation of endothelium caused coronary capillary endothelial swelling and pseudopod formation. Thus increased coronary neutrophil transit time and retention are due to structural changes of coronary endothelial cells or other effects of the inflammatory response occurring within coronary capillaries, not only due to activation of leukocytes.

Animals↗

PubMed2003

Structure-function studies of human apolipoprotein A-V: a regulator of plasma lipid homeostasis.

To investigate structure and function relations of a new member of the exchangeable apolipoprotein family that modulates plasma lipid levels, recombinant human apolipoprotein (apo) A-V was produced in Escherichia coli and isolated by a combination of nickel chelation affinity chromatography and reversed-phase HPLC. Antibodies directed against apoA-V were generated and employed in immunoblotting experiments. Anti-apoA-V IgG gave a strong response against recombinant apoA-V from E. coli and human apoA-V expressed in transgenic mice, but did not recognize human apoA-I or apoA-IV. In neutral-pH buffers, at concentrations of >0.1 mg/mL, isolated lipid-free apoA-V is poorly soluble. By contrast, apoA-V is soluble in 50 mM sodium citrate (pH 3.0). Far-UV circular dichroism analysis and spectral deconvolution reveal that apoA-V possesses 32% alpha-helix, 33% beta-sheet, 16% beta-turn, and 18% random coil secondary structure conformers. Temperature-induced denaturation studies gave rise to a transition midpoint of 47.1 degrees C. Upon being cooled to ambient temperature from 85 degrees C, apoA-V failed to recover all of the negative ellipticity present in unheated apoA-V. ApoA-V interacts with bilayer vesicles of dimyristoylphosphatidylcholine to form discoidal complexes with diameters in the range of 15-20 nm. However, apoA-V was a poor activator of lecithin:cholesterol acyltransferase where the activity was 8.5 +/- 1.8% of that of apoA-I. Furthermore, apoA-V failed to support enhanced efflux of cholesterol from cAMP-treated J774 macrophages, although low levels of efflux were obtained from unstimulated cells. Taken together, the results demonstrate recombinant apoA-V possesses unique structural and functional characteristics, in keeping with its proposed role in the modulation of plasma lipid levels.

Animals↗

PubMed1996

Phospholipases: structural and functional motifs for working at an interface.

Phospholipases form a ubiquitous class of enzymes optimized to catalyze the hydrolysis of phospholipids. Because their products are often second messengers, they are highly regulated by the cell. For a given ester bond, there are separate secreted as well as cytoplasmic phospholipases with different substrate specificities and modes of regulation. As it becomes available, structural information provides a view of interfacial catalysis for several of these phospholipases on a molecular level. Recent structural advances include solution structures of a pancreatic phospholipase A2 in the absence and presence of a micellar interface, crystal structures of a bacterial phosphatidylinositol-phospholipase C whose active site is reminiscent of ribonuclease, and a Ca2+ lipid binding domain with high homology to regions in several cytoplasmic phospholipases that can model the way those proteins interact with the membrane surface. Phospholipases also have a wide and complex array of regulatory mechanisms involving cytoplasmic proteins, notably G-proteins, as well as different effector lipids (e.g., phosphatidylinositol-4,5-biphosphate, or PIP2) or Ca2+. Deconvolution of these interactions is necessary to understand their roles in different signal transduction pathways.-Roberts, M. F. Phospholipases: structural and functional motifs for working at an interface.

Animals↗

PubMed1996

Abnormalities of insulin pulsatility and glucose oscillations during meals in obese noninsulin-dependent diabetic patients: effects of weight reduction.

Twenty-seven obese patients, including 8 with normal glucose tolerance, 10 with subclinical NIDDM, and 9 with overt noninsulin-dependent diabetes mellitus (NIDDM), were studied before and after prolonged weight loss to assess the effects of the underlying defects of diabetes per se from those of obesity and chronic hyperglycemia on the regulation of pulsatile insulin secretion. Serial measurements of insulin secretion and plasma glucose were obtained during 3 standardized mixed meals consumed over 12 h. Insulin secretion rates were calculated by deconvoluting plasma C peptide levels using a mathematical model for C peptide clearance and kinetic parameters derived individually in each subject. Absolute (nadir to peak) and relative (fold increase above nadir) amplitudes of each insulin secretory pulse and glucose oscillation were calculated. Compared to the obese controls, the subclinical and overt NIDDM patients manifested the following abnormal responses: 1) decreased relative amplitudes of insulin pulses, 2) reduced frequency of glucose oscillations, 3) increased absolute amplitudes of glucose oscillations, 4) decreased temporal concomitance between peaks of insulin pulses and glucose oscillations, 5) reduced correlation between the relative amplitudes of glucose oscillations concomitant with insulin pulses, and 6) temporal disorganization of the insulin pulse profiles. These defects were more severe in the overt NIDDM patients, and weight loss only partially reversed these abnormalities in both NIDDM groups. These findings indicate that beta-cell responsiveness is reduced, and the regulation of insulin secretion is abnormal under physiological conditions in all patients with NIDDM, including those without clinical manifestations of the disease. These abnormalities are not completely normalized with weight loss, even in patients who achieve metabolic control comparable to that in obese controls. The results are consistent with the presence of an inherent beta-cell defect that contributes to secretory derangements in subclinical NIDDM patients. This abnormality precedes frank hyperglycemia and may ultimately contribute to the development of overt NIDDM.

Blood Glucose↗

PubMed2005

Orthogonal chemical genetic approaches for unraveling signaling pathways.

While chemical genetic approach uses small molecules to probe protein functions in cells or organisms, orthogonal chemical genetics refers to strategies that utilize reengineered protein-small molecule interfaces, to alter specificities, in order to probe their functions. The advantage of orthogonal chemical genetics is that the changes at the interfaces are generally so minute that it goes undetected by natural processes, and thus depicts a true physiological picture of biological phenomenon. This review highlights the recent advances in the area of orthogonal chemical genetics, especially those designed to probe signaling processes. Dynamic protein-protein and enzyme-substrate interactions following stimuli form the foundation of signal transduction. These processes not only break spatial and temporal boundaries between interacting proteins, but also impart distinct regulatory properties by creating functional diversity at the interfaces. Functional and temporal modulation of these dynamic interactions by specific chemical probes provides extremely powerful tools to initiate, ablate, decouple and deconvolute different components of a signaling pathway at multiple stages. Not surprisingly, multiple receptor-ligand reengineering approaches have been developed in the last decade to selectively manipulate these transient interactions with the aim of unraveling signaling events. However, given the diversity of protein-protein interactions and novel chemical genetic probes developed to perturb these processes, a short review cannot do adequate justice to all aspects of signaling. For this reason, this review focuses on some orthogonal chemical-genetic strategies that are developed to study signaling processes involving enzyme-substrate interactions.

Adenosine Triphosphate↗

PubMed2002

Combinatorial selection of RNA ligands for complex cellular targets : the RNA liagands-based proteomics.

This study explores the selection of high affinity RNA ligands for the complex cellular targets present in crude HeLa nuclear extract through directed evolution and deconvolution. RNA ligands for the mixed nuclear targets were selected from around 6 x 10(14) RNA sequences through an iterated enrichment process. RNA ligands for various gene products of the extract were simultaneously selected and were shown to specifically interact with their target molecules. The target molecules were isolated from the nuclear extract by affinity chromatography using columns tagged with the RNA ligands, resolved on two-dimensional gels, and identified by mass spectrometry. These RNA ligands may be useful in characterizing novel functions of cellular proteins and modulating complex molecular events.

Base Sequence↗

PubMed1993

Lack of control by glucose of ultradian insulin secretory oscillations in impaired glucose tolerance and in non-insulin-dependent diabetes mellitus.

Normal subjects demonstrate the presence of ultradian oscillations (period 80-150 min) in insulin secretion rate (ISR) tightly coupled to glucose oscillations of similar period. These oscillations appear to be a function of the feedback loop linking glucose and insulin. The present study was undertaken to determine whether the control by glucose of the ultradian oscillations in insulin secretion is altered in impaired glucose tolerance IGT and in non-insulin-dependent diabetes mellitus (NIDDM). Patients with NIDDM (n = 7), IGT (n = 4), and matched nondiabetic controls (n = 5) were studied under three separate protocols that involved administration of glucose at either a constant rate of 6 mg/kg per min for 28 h or in one of two oscillatory patterns at the same overall mean rate. The amplitude of the oscillations was 33% above and below the mean infusion rate, and their respective periods were 144 min (slow oscillatory infusion) or 96 min (rapid oscillatory infusion). Insulin, C-peptide, and glucose were sampled at 10-min intervals during the last 24 h of each study. ISRs were calculated by deconvolution of C-peptide levels. Analysis of the data showed that (a) the tight temporal coupling between glucose and ISR in the nondiabetic controls was impaired in the IGT and NIDDM groups as demonstrated by pulse analysis, cross-correlation analysis, and spectral analysis; (b) the absolute amplitude of the ISR pulses progressively declined with the transition from obesity to IGT to NIDDM; and (c) the absolute amplitude of the ISR oscillations failed to increase appropriately with increasing absolute amplitude of glucose oscillations in the IGT and NIDDM subjects compared with the control group. In conclusion, the present study demonstrates that important dynamic properties of the feedback loop linking insulin secretion and glucose are disrupted not only in established NIDDM but also in conditions where glucose tolerance is only minimally impaired. Further studies are needed to determine how early in the course of beta-cell dysfunction this lack of control by glucose of the ultradian oscillations in insulin secretion occurs and to define more precisely if this phenomenon plays a pathogenetic role in the onset of hyperglycemia in genetically susceptible individuals.

C-Peptide↗

PubMed2002

The molecular architecture of calcium microdomains in rat cardiomyocytes.

We have used standard indirect immunofluorescence techniques in combination with wide-field microscopy and image deconvolution to assess the distribution of proteins implicated in excitation-contraction coupling and Ca(2+) homeostasis in adult rat cardiomyocytes. We begin by discussing our earlier results and summarizing what is known about the molecular architecture of this species to provide a rationale for the work presented here. The previous results showed that the dyads contain Ca(2+) channels and ryanodine receptors, but few Na(+) channels or Na(+)/Ca(2+) exchangers. The latter proteins were not colocalized elsewhere on the membrane, and we have now found that they appear to be minimally associated with caveolin-3. None of the molecules examined are distributed uniformly in the membranes in which they are located but are organized into discrete clusters attached to the underlying cytoskeleton, an arrangement that, at the level of light microscopy, does not appear to be affected by the enzymatic dissociation used to study single cells. Analysis of how the clusters are organized and distributed throughout the volume of the cell suggests that there may be differences in excitation-contraction coupling between the cell surface and the interior.

Animals↗

PubMed1996

Effects of prolonged glucose infusion on insulin secretion, clearance, and action in normal subjects.

It was the aim of this study to determine whether prolonged hyperglycemia can produce "glucose toxicity" in normal human subjects. To this end, plasma glucose was clamped at approximately 5, approximately 8.8, and approximately 12.6 mM for 68 h in healthy volunteers. Rates of insulin secretion (by deconvolution of plasma C-peptide) and rates of insulin clearance [area under curve (AUC) 24 h insulin secretion/AUC 24 h insulin] were determined. Pre- and posthyperglycemia glucose turnover was measured (with [6,6-2H2]glucose) during euglycemic-hyperinsulinemic clamping to assess peripheral (muscle) and hepatic insulin action. Hyperglycemia (approximately 12.6 mM) for 68 h was associated with significant reductions in rates of insulin secretion (-35%, P < 0.05), insulin clearance (-57%, P < 0.05), glucose infusion rates needed to maintain hyperglycemia (-36%, P < 0.05), and insulin-stimulated glucose uptake (-55%, P < 0.01). No significant changes were seen during approximately 8.8 mM hyperglycemia or during euglycemia. These data showed that 12.6 mM hyperglycemia, but not 8.8 mM hyperglycemia or euglycemia, was associated with reduced insulin secretion, insulin clearance, and peripheral (muscle) insulin action. We concluded that 1) in normal subjects, desensitization to glucose involving beta-cells and muscle developed at plasma glucose concentrations between approximately 9 and approximately 12 mM, and 2) these effects were partially compensated for by a decrease in insulin clearance.

Adult↗