bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of specific activity of labeled ligands by nonlinear regression analysis
Authors: Durr J. A.
Published: December 2017

The specific radioactivity (SA) of 125I-lysine vasopressin (LVP) was determined by analyzing the binding B (cpm/tube) of variable amounts of tracer T (cpm/tube) to a constant amount of an LVP antibody, in the presence of known quantities L (mol/tube) of LVP standards. The parameters of the equations B = f(F) and B/F = g(T), describing B as a function of free F (cpm/tube) tracer or the ratios B/F as a function of T, were first calculated by nonlinear regression analysis of the results obtained with tracer alone. Then the dependent variables B or B/F were measured in the presence of LVP and analyzed with the same equations by substituting the independent variables F or T with (F + alpha FL) and (T + alpha L), respectively, where alpha (cpm/mol) represents a measure of the SA and FL (FL = L.F/T), free LVP, respectively. The SA was thus treated as an unknown parameter to be calculated by nonlinear regression. This method was compared with the traditional interpolation of the SA from the self-displacement and standard curves. Tracer and ligand were found to have the same affinity for the binding sites, since the set of equations B = f(F + alpha FL) and B/F = g(T + alpha L), describing the binding of the tracer in the presence of LVP and equations B = f(F) and B/F = g(T) to which these equations reduce in the absence of LVP (L = 0), had identical binding parameters. To be valid, any method based on self-displacement requires that the tracer and standards have the same affinity for the binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Differential effects of corticosteroids on rat peripheral blood T-lymphocyte mitogenesis in vivo and in vitro
Authors: Wiegers G. J. , Croiset G. , Reul J. M. , Holsboer F. , de Kloet E. R.
Published: December 2017

The effects of corticosteroids were studied on the concanavalin A (Con A)-induced mitogenesis of peripheral blood T-lymphocytes obtained from intact and adrenalectomized (ADX) Wistar rats. One week of adrenalectomy reduced the proliferative response of T-cells by 65% compared with sham-operated controls. Substitution of ADX rats with subcutaneously implanted 12.5-mg corticosterone (Cort) pellets, which resulted in low circulating Cort levels (17 +/- 3 nM), restored the reduced proliferative capacity to that of sham-ADX animals. In contrast, T-lymphocyte proliferation was nearly absent in ADX rats substituted with high circulating Cort levels (173 +/- 15 nM; 100-mg Cort pellet). In vitro, Cort suppressed the mitogenic response of T-lymphocytes from ADX and sham-ADX animals. The glucocorticoid antagonist RU-486 (500 nM) completely blocked this suppressive effect. However, a 10 times lower concentration of RU-486 reversed the effects of a low (10 nM) Cort concentration from suppression to stimulation. It is concluded that high Cort concentrations in vivo and in vitro suppressed T-lymphocyte mitogenesis but that low concentrations in vivo were stimulatory, whereas this stimulation in vitro occurred only in the presence of antiglucocorticoids. These opposing effects of Cort emphasize a bimodal regulatory role of this hormone in immune regulation that may be mediated by different corticosteroid receptors.
Temporal changes in insulin resistance and secretion in 24-h-fasted conscious pregnant rats
Authors: Rossi G. , Sherwin R. S. , Penzias A. S. , Lapaczewski P. , Jacob R. J. , Shulman G. I. , Diamond M. P.
Published: December 2017

To determine the temporal sequence of pregnancy-induced changes in insulin action and secretion, awake midpregnant (11-12 days) and late pregnant (19-20 days) rats underwent a two-step euglycemic hyperinsulinemic or a hyperglycemic clamp study after a 24-h fast. During euglycemia, insulin-stimulated increments in glucose uptake and clearance in midpregnant rats were reduced by 60-70% at the lower dose (insulin approximately 360 pM) and by 20-30% at the higher dose (insulin approximately 1,750 pM; P < 0.01 vs. virgin controls). Insulin action was also diminished in late pregnant rats. However, the magnitude of resistance did not increase. Insulin-mediated suppression of glucose production was only minimally impaired in midpregnancy. In contrast, glucose production was virtually unchanged in late pregnancy, even at the highest insulin dose. During hyperglycemia, insulin responses in late pregnancy were markedly increased 5-fold above controls and 2.5-fold above midpregnant rats (P < 0.05). We conclude that rat pregnancy is characterized by the early appearance of peripheral insulin resistance. As pregnancy progresses toward term, marked hepatic insulin resistance and insulin hypersecretion develop, whereas peripheral insulin resistance demonstrates negligible changes. These data imply that insulin hypersecretion during late pregnancy is most closely linked to hepatic insulin resistance, at least in 24-h-fasted animals.
Mammalian bombesin as a hormone in ovine pregnancy: ontogeny, origin, and molecular forms
Authors: Giraud A. , Parker L. , Taupin D. , Hardy K. , Shulkes A.
Published: December 2017

Mammalian gastrin-releasing peptides (GRP) are present in female reproductive tissues and stimulate uterine contraction and DNA synthesis in the endometrium. We set out to establish whether the GRP were likely to play a role in fetal development by measuring the fetal and maternal plasma concentrations in chronically cannulated fetal sheep from 115 days gestation to term (145 days) and for 18 days after parturition. Placental fluids and fetal urine were also obtained. In a separate series of animals, uterine, placental, and fetal gut and lung tissues were collected. Samples were extracted in acid and assayed by radioimmunoassay, and molecular forms were characterized by high-performance liquid chromatography. GRP were present in the decidua (> 10 pmol/g), as well as placental, uterine, lung, and gastrointestinal tissues (all < 6 pmol/g). Fetal and maternal plasma GRP were elevated compared with nonpregnant ewes, falling sharply after parturition. Placental fluids and urine also contained GRP (80-410 fmol/ml). The main molecular form in all tissues and fluids examined coeluted with porcine GRP-(18-27). GRP immunoreactivity was primarily localized to epithelial cells of the decidua. These data suggest that plasma GRP is probably derived from the decidua and may play a role as a circulating hormone in ovine fetal and uterine development.
GLUT-4 content in plasma membrane of muscle from patients with non-insulin-dependent diabetes mellitus
Authors: Lund S. , Vestergaard H. , Andersen P. H. , Schmitz O. , Gotzsche L. B. , Pedersen O.
Published: December 2017

The abundance of GLUT-4 protein in both total crude membrane and plasma membrane fractions of vastus lateralis muscle from 13 obese non-insulin-dependent diabetes mellitus (NIDDM) patients and 14 healthy subjects were examined in the fasting state and after supraphysiological hyperinsulinemia. In the basal state the immunoreactive mass of GLUT-4 protein both in the crude membrane preparation and in the plasma membrane fraction was similar in NIDDM patients and control subjects. Moreover, in vivo insulin exposure neither for 30 min nor for 4 h had any impact on the content of GLUT-4 protein in plasma membranes. With the use of the same methodology, antibody, and achieving the same degree of plasma membrane purification and recovery, we found, however, that intraperitoneal administration of insulin to 7-wk-old rats within 30 min increased the content of GLUT-4 protein more than twofold (P < 0.01) in the plasma membrane from red gastrocnemius and soleus muscle. In conclusion, when the subcellular fractionation method was applied to human muscle biopsies taken in the basal state, no difference could be found in the plasma membrane content of immunoreactive GLUT-4 protein between NIDDM patients and normal subjects. With this technique, we were unable to show evidence for a regulatory effect of insulin on the plasma membrane level of GLUT-4 protein in human muscle.
Phosphate transport by osteoblasts from X-linked hypophosphatemic mice
Authors: Ecarot B. , Caverzasio J. , Desbarats M. , Bonjour J. P. , Glorieux F. H.
Published: December 2017

Hypophosphatemic vitamin D-resistant rickets is characterized by impaired renal reabsorption of Pi. The underlying mechanism of this abnormality remains unknown. Because the osteoblast is likely a target for the HYP mutation, we investigated the Pi transport activity in osteoblasts isolated from the murine homologue for the human disease, the Hyp mouse. Kinetic analysis of sodium-dependent Pi uptake in quiescent normal and Hyp osteoblasts indicated no significant differences in apparent maximal capacity (Vmax) and apparent affinity (Km) of the carrier for Pi. In rapidly growing cells, higher levels of Pi uptake were observed in mutants cells associated with a 1.4- to 1.7-fold increase in Vmax and no change in Km for Pi. This increase in Pi uptake seemed related to changes in the sodium electrochemical gradient inasmuch as a similar increase was observed in alanine uptake. The adaptive response of sodium-dependent Pi transport to Pi deprivation was not altered in mutant cells relative to normal cells. To test whether the expression of a Pi transport defect was dependent on a humoral factor for its expression, we evaluated the activity of the serum from Hyp mice on Pi transport in osteoblasts from both genotypes. No difference in activity was observed between sera from normal and mutant mice. In summary, cultured osteoblasts derived from Hyp mice did not express impaired sodium-dependent Pi transport when compared with cells from normal mice.
Interleukin-1 increases protein kinase A activity by a cAMP-independent mechanism in AtT-20 cells
Authors: Gwosdow A. R. , O'Connell N. A. , Abou-Samra A. B.
Published: December 2017

A recent study from this laboratory has shown that the inflammatory mediator, interleukin-1 alpha (IL-1 alpha), stimulates protein kinase A (PKA) activity and adrenocorticotropic hormone (ACTH) secretion from AtT-20 cells without any detectable increase in intracellular cAMP accumulation. The present studies were conducted to determine if cAMP is involved in IL-1 alpha activation of PKA and if PKA is responsible for IL-1 alpha-induced ACTH release from AtT-20 cells. The data are consistent with a novel mechanism of PKA activation that does not involve cAMP. Inhibition of adenylate cyclase with 2'5'-dideoxyadenosine (2'5'-DDA) did not affect IL-1 alpha-induced increases in PKA activity and ACTH secretion. In contrast, CRF-stimulated PKA activity and ACTH secretion were inhibited by 2'5'-DDA. Additional evidence was obtained using the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). IBMX did not alter IL-1 alpha-induced PKA activity or ACTH secretion, yet IBMX potentiated CRF-induced cAMP accumulation. Inhibition of PKA with the PKA inhibitor, H-8, blocked activation of PKA and ACTH secretion by both IL-1 alpha and CRF in AtT-20 cells. These observations demonstrate that 1) the mechanism of IL-1 alpha activation of PKA is independent of adenylate cyclase or cAMP and 2) PKA is used by IL-1 alpha to induce ACTH secretion from AtT-20 cells.
Effect of renal nerves on expression of renin and angiotensinogen genes in rat kidneys
Authors: Nakamura A. , Johns E. J.
Published: December 2017

In this study, we try to determine the influence of renal nerve activity on renal function, plasma renin activity (PRA), and the corresponding expression of renin and angiotensinogen genes in the kidney. In pentobarbitone-anesthetized rats, the left renal nerves were stimulated (15 V, 0.2 ms) at frequencies to reduce left renal blood flow by 15, 30, and 45%. There were corresponding reductions in glomerular filtration rate from 12 to 52% and absolute and fractional sodium excretions from 20 to 75%. PRA levels in control rats were 10.8 +/- 1.5 and were increased to 65.9 +/- 9.1, 144.2 +/- 19.7, and 277.2 +/- 22.0 ng angiotensin I.h-1.ml-1 after 1 h at each of the three levels of nerve stimulation. Renal renin mRNA was similar in innervated and denervated kidneys and was not affected by the lowest level of nerve stimulation; however, neurally induced decreases in blood flow to 30 and 45% increased renin mRNA levels by 3.0- and 3.4-fold (both P < 0.05), respectively. Angiotensinogen mRNA levels were higher (P < 0.05) in kidneys subjected to the lowest level of nerve stimulation, but when renal blood flow was reduced by 30 and 45%, expression of this gene was unchanged. Stimulation of the renal nerves in the presence of the beta 1-adrenoceptor antagonist atenolol only doubled PRA at the highest rates of stimulation. Neither renal renin nor angiotensinogen mRNA were changed during neurally mediated reductions in renal blood flow of 15 or 30% after administration of atenolol.(ABSTRACT TRUNCATED AT 250 WORDS)
Reliability of error estimates from the minimal model: implications for measurements in physiological studies
Authors: Prigeon R. L. , Kahn S. E. , Porte D.
Published: December 2017

MINMOD provides an estimate of the error in the insulin sensitivity index (SI) and glucose effectiveness at basal insulin (Sg) as the fractional standard deviation (FSD). The validity of the FSD estimate has not been assessed in a large number of human studies, nor has a comparison of the accuracies achievable using the two different intravenous glucose tolerance test (IVGTT) protocols (glucose only and tolbutamide protocol) been performed. To address these two issues, we obtained the FSD value and performed Monte Carlo simulations for 237 IVGTT studies. The FSD underestimated the true error as determined as the coefficient of variation from Monte Carlo simulation (COV-MC) with the ratio of COV-MC to FSD being 3.07 +/- 0.20 (mean +/- SE) for SI using the tolbutamide protocol. Additionally, the mean COV-MC for glucose-only protocol was approximately two to three times that for the tolbutamide protocol for both SI and Sg. We conclude that FSD underestimates the true error in SI and Sg. Additionally, more accurate results are obtained from the tolbutamide protocol than with the glucose-only protocol.
Measurement of tissue protein synthesis rates in vivo: a critical analysis of contrasting methods
Authors: Garlick P. J. , McNurlan M. A. , Essen P. , Wernerman J.
Published: December 2017

The extension of the flooding method for measuring the rate of protein synthesis, from animal to human tissues, has led to criticism. This is based on the observation that in human muscle, unlike animal tissues, the rate of synthesis in the fasting state measured with constant infusion is lower than that obtained with the flooding technique. Moreover, incorporation of infused tracer can be enhanced with a simultaneous flood, although an inhibition of incorporation has also been reported. Explanations for these observed discrepancies are explored. Evidence from studies in human muscle both with flooding and with a nonisotopic technique have given no indication of a stimulation of protein synthesis during flooding. It is therefore concluded that the most likely explanation for the discrepancy between methods is that changes in the isotopic enrichment of the precursor amino acid, which are minimized by the flooding procedure, are not adequately accounted for with the constant infusion method.
Prediction of positional isotopomers of the citric acid cycle: the syntactic approach
Authors: Cohen D. M. , Bergman R. N.
Published: December 2017

We propose a syntactic approach to modeling of biochemical fluxes that combines a rule-based description of atomic transfer in chemical reactions with a structurally oriented, stochastic model of chemical reaction kinetics. This approach avoids the use of differential equations to describe the production and disappearance of each molecule. The computer simulation predicts the changes over time in the abundance of each positional isotopomer of every metabolic intermediate in the citric acid cycle of heart cells, subsequent to administration of [2-13C]acetate (including natural abundance of 13C). (Positional isotopomers are isomers that differ in the positions of isotopes within the molecule.) The 32 positional isotopomers of glutamate fell into four groups with similar intragroup dynamics but with very different amplitudes. From the relative abundance of each isotopomer of glutamate, we calculate the relative area of multiplets of the nuclear magnetic resonance spectrum.
Nutritional influence on the recovery of 14CO2 in critically ill trauma patients
Authors: Jeevanandam M. , Holaday N. J. , Petersen S. R.
Published: December 2017

Several factors, including age, nutritional and metabolic status, and underlying pathological conditions, should be considered in estimating the correction factor for labeled CO2 retention. We have evaluated the nutritional influence of this correction factor in a group of severely injured (injury severity score = 33 +/- 5), hypermetabolic, and highly catabolic adult trauma patients. Primed-constant infusion of NaH14CO3 was used, and the breath 14CO2 radioactivity was measured. The experiment was conducted once in the fasting condition during the early catabolic "flow" phase of injury and again during and after a week of intravenous feeding. The mean value of the 14CO2 recovery (85.2 +/- 2.7%) in the basal fasting condition was higher than the value of 81% commonly used in normal subjects. During intravenous nutritional support the recovery was 100.8 +/- 1.7%. Recovery of tracer increased linearly with O2 uptake and CO2 production. The linear expression obtained between recovery and CO2 production could estimate the recovery within 3% in trauma patients, and that obtained by multiple regression with CO2 production and O2 uptake would predict the recovery within 1.5%.
Use of 2H2O to study labeling in plasma glucose and hepatic glycogen during a hyperglycemic clamp
Authors: Shalwitz R. A. , Beth T. J. , MacLeod A. M. , Tucker S. J. , Rolison G. G.
Published: December 2017

In this study, we demonstrate the use of 2H2O, in a manner analogous to 3H2O, to study gluconeogenic flux (deuterium labeling at the carbon-6 position of glucose) relative to overall flux through glucose 6-phosphate (deuterium labeling at the carbon-2 position of glucose) into glucose output and glycogen synthesis during hyperglycemia. Before the study (4 days), jugular and carotid catheters were placed. Rats were fasted for 17 h before the study. 2H2O was infused for 2 h at 3 ml/h, with a subsequent 1-h equilibration period. A hyperglycemic clamp at 180 mg/dl (10 mM) was then performed for 90 min (plasma samples obtained at 10-min intervals). At the end of the experiment, anesthesia was induced and the liver removed. Gas chromatography-mass spectroscopy isotopomer analysis of four different mass clusters from glucose was used to determine deuterium enrichment on the carbon-2 (E2D) and carbon-6 (E6D) positions of plasma glucose and glycogen-glucose. The results show that the labeling pattern in glycogen and plasma glucose was virtually identical. In addition, the E6D-to-E2D ratio in plasma glucose did not change during hyperglycemia. Additional studies were performed to show that the E6D-to-E2D ratio was decreased in the fed state and that the fed animal, compared with the fasted rat, had a marked increase in the ratio when given an epinephrine infusion. Thus it was concluded that this was a robust new technique for analyzing glucose and glycogen metabolism in rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Excretion of IL-6 by astronauts during spaceflight
Authors: Stein T. P. , Schluter M. D.
Published: December 2017

Ascent to and living under the microgravity conditions found during spaceflight is an unfamiliar environment for humans. The adaptation to the space environment may be perceived by the body as a stress. On the ground, stress results in increased cytokine activity. The objective of this study was to determine whether spaceflight is associated with increased cytokine activity. The mean daily urinary interleukin-6 (IL-6) excretion rate was measured on 24-h urine pools collected from four payload crew members from 11 days before launch to 7 days after landing for a total of 27 days. In addition, in-flight data were obtained from two orbiter crew members. The experiment was conducted before, during, and after the 1991 9.5-day SLS-1 (Columbia) space shuttle mission. Dietary intake and urine output were monitored continuously for the 27-day period for the four payload crew. Results are as follows: 1) urinary IL-6 excretion and cortisol excretion were increased on the 1st day of spaceflight, suggesting an acute-phase response; 2) elevated levels of IL-6 were not found in the urine on any other days before or during flight; and 3) two of the subjects had markedly increased IL-6 excretion rates after landing.
Angiotensin II in brown adipose tissue from young and adult Zucker obese and lean rats
Authors: Cassis L. A.
Published: December 2017

Previous studies demonstrated that interscapular brown adipose tissue (ISBAT) produces angiotensin II (ANG II), which facilitates sympathetic neurotransmission (SN). ANG II content and regulation of SN were examined in young (17 days) and adult (16 wk) Zucker obese and lean rats. ANG II content in ISBAT from preobese rats was decreased compared with lean littermates. Evoked 3H overflow in ISBAT slices preloaded with [3H]NE was greater in preobese rats compared with control. ANG II increased evoked 3H overflow in ISBAT slices to a greater extent in preobese rats compared with control. [3H]NE uptake in ISBAT slices from preobese rats was decreased compared with control. In adult obese rats, plasma renin activity was decreased compared with control. ISBAT ANG II content was increased in adult obese rats compared with control. Evoked 3H overflow in ISBAT slices preloaded with [3H]NE was not different between obese and control. ANG II did not increase evoked 3H overflow in obese rats; however, ANG II increased evoked 3H overflow in lean rats. [3H]NE uptake in ISBAT slices from obese rats was decreased compared with control. These results suggest that ANG II modulation of SN activity is decreased in ISBAT from adult obese rats. In contrast, in young obese rats, increased SN activity and ANG II regulation of SN were evident in brown adipose tissue.
Dietary restriction increases insulin sensitivity and lowers blood glucose in rhesus monkeys
Authors: Kemnitz J. W. , Roecker E. B. , Weindruch R. , Elson D. F. , Baum S. T. , Bergman R. N.
Published: December 2017

Insulin sensitivity and glucose tolerance typically decline during later life. In a multidimensional randomized trial of the effects of dietary restriction started in adulthood on the processes of aging, we are studying insulin sensitivity and glucoregulation longitudinally in control (C, n = 15, fed a defined diet ad libitum for 6–8 h/day) and restricted (R, n = 15, fed 30% less than C) monkeys using the Modified Minimal Model method. Linear rates of change were calculated for individual animals through 30 mo of diet treatment and compared between treatment groups. Basal glucose, basal insulin, and insulin responses to glucose and tolbutamide increased for C and decreased for R animals (P < or = 0.002), whereas insulin sensitivity decreased for C and increased for R (P = 0.008). Glycosylated hemoglobin at 30 mo was marginally lower in R (P = 0.06) and was positively correlated with fasting plasma glucose (r = 0.508, P < 0.001). Insulin changes were significantly correlated with changes in adiposity (weight and abdominal circumference). Identification of the mechanisms through which these effects are achieved may aid in ameliorating glucose intolerance, insulin resistance, and associated illnesses in older persons.
Fat metabolism in human obesity
Authors: Campbell P. J. , Carlson M. G. , Nurjhan N.
Published: December 2017

Excessive fat turnover and oxidation might cause the insulin resistance of carbohydrate metabolism in obese humans. We studied the response of free fatty acid (FFA) metabolism in lean and obese volunteers to sequential insulin infusions of 4, 8, 25, and 400 mU.m-2.min-1. The insulin dose-response curves for suppression of FFA concentration, FFA turnover ([1-14C]palmitate), and lipolysis ([2H5]glycerol) were shifted to the right in the obese subjects (insulin concentrations that produced a half-maximal response, lean vs. obese: 103 +/- 21 vs. 273 +/- 41, 96 +/- 11 vs. 264 +/- 44, and 101 +/- 23 vs. 266 +/- 44 pM, all P < 0.05), consistent with insulin resistance of FFA metabolism in obesity. After the overnight fast, FFA turnover per fat mass was decreased in obese subjects (37 +/- 4 vs. 20 +/- 3 mumol.kg fat mass-1.min-1, P < 0.01) as the result of suppression of lipolysis by the hyperinsulinemia of obesity and an increased fractional reesterification of FFA before leaving the adipocyte (primary FFA reesterification; 0.14 +/- 0.03 vs. 0.35 +/- 0.06, P < 0.05). Nevertheless, FFA turnover per fat-free mass (FFM) was also greater in the obese volunteers (8.5 +/- 0.7 vs. 11.0 +/- 1.0 mumol.kg FFM-1.min-1, P < 0.05) but only as the result of increased reesterification of intravascular FFA (secondary reesterification; 1.8 +/- 0.5 vs. 4.8 +/- 1.1 mumol.kg FFM-1.min-1, P < 0.01), since FFA oxidation was the same in the two groups throughout the insulin dose-response curve.(ABSTRACT TRUNCATED AT 250 WORDS)
Impaired beta-adrenergic receptor-mediated regulation of gene expression in adipocytes from older rats
Authors: Shilo L. , Chin J. H. , Hoffman B. B.
Published: December 2017

With aging, in many cells there is a blunted capacity of beta-adrenergic agonists to increase adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. We wondered if blunted cAMP responses led to impaired regulation of genes in which expression is modified by cAMP. To test this hypothesis, adipocytes were isolated from 2- and 12- to 16-mo-old Sprague-Dawley rats that were stimulated with isoproterenol, and the inhibitory effect on expression of genes for lipoprotein lipase (LPL) and fatty acid synthase (FAS) were examined. Isoproterenol-stimulated cAMP accumulation and glycerol release were impaired in adipocytes from the older rats. Isoproterenol inhibited expression of LPL and FAS mRNAs in cells from the younger but not older rats. Removal of endogenous adenosine partially restored isoproterenol-mediated inhibition of expression of these genes in the cells. These results demonstrate that blunted cAMP responses associated with aging in obese Sprague-Dawley rats led to altered regulation of LPL and FAS genes. These findings extend the possible physiological implications of impaired hormone-induced cAMP responses with aging.
A general model for analysis of the tricarboxylic acid cycle with use of [13C]glutamate isotopomer measurements
Authors: Vogt J. A. , Fischman A. J. , Kempf M. , Yu Y. M. , Tompkins R. G. , Burke J. F.
Published: December 2017

A generalized steady-state model was developed for determining tricarboxylic acid cycle fractional fluxes from 13C nuclear magnetic resonance (NMR) data. The model relates the measured mole fractions of [13C]glutamate isotopomers to the fractional fluxes and predicted mole fractions of isotopomers of oxaloacetate (OAA) and acetyl-CoA. This model includes cycling between OAA and fumarate. Fractional fluxes are determined by fitting the model equations to NMR parameters by use of nonlinear least squares. Although only fractional fluxes can be determined from 13C-NMR data, when they are combined with mass spectroscopic measurements, absolute values can be derived. A specific metabolic system represented by published 13C-NMR data from extracts of hearts perfused with [13C]acetate, [13C]pyruvate (PYR), and [13C]acetate plus [13C]PYR was used to test the model. The intensities of predicted 13C-NMR splitting patterns were compared with observed values, and there was excellent agreement between observed and predicted signal intensities. With this model, important physiological parameters, including the OAA-derived fraction of inflow to PYR, PYR-derived fraction of inflow to acetyl-CoA, citrate-derived fraction of inflow to OAA, and PYR-derived fraction of inflow to OAA, can be determined.
Lipoprotein lipase and hormone-sensitive lipase activity and mRNA in rat adipose tissue during pregnancy
Authors: Martin-Hidalgo A. , Holm C. , Belfrage P. , Schotz M. C. , Herrera E.
Published: December 2017

To investigate the factors controlling maternal depot fat accumulation during early pregnancy and net decrease during late pregnancy, the activity and mRNA expression of adipose tissue lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) were related to several other lipid metabolic parameters. Virgin control rats, pregnant rats (at days 12, 15, 19, and 21), and lactating rats (at days 5 and 10 postpartum) were studied. In adipose lumbar tissue of late pregnant rats, LPL activity decreased to about one-third that of the virgin control animals, with < 10% of initial LPL mRNA expressed as determined by Northern blots. HSL activity increased maximally 1.5-fold with a fourfold increase of HSL expression at days 12-15 of pregnancy and decreased to control levels after parturition. The HSL-to-LPL mRNA and activity ratios were enhanced from days 15 and 19 of pregnancy, respectively, and remained so even during lactation, mainly because of the marked lowering of the LPL values. This enhancement coincided with increments in plasma free fatty acids and glycerol levels indicating an increased depot fat breakdown. These results give no indication of an involvement of LPL and HSL gene expression changes in the accumulation of maternal depot during early pregnancy. In contrast, such changes could be responsible for the net breakdown of this fat depot during late gestation. Thus, during this physiological state, long-term (e.g., transcriptional) regulation of LPL and HSL gene expression could be an important mechanism for the control of adipose tissue mass breakdown during late gestation.
GLUT-1 and GLUT-2 mRNA, protein, and glucose transporter activity in cultured fetal and adult hepatocytes
Authors: Levitsky L. L. , Zheng Q. , Mink K. , Rhoads D. B.
Published: December 2017

To understand glycogenesis in the fetal hepatocyte, we examined glucose transport in cultured fetal and adult male rat hepatocytes. GLUT-1 mRNA was detected in fetal hepatocytes at isolation but in adult hepatocytes only after culture. GLUT-1 mRNA was more abundant in fetal than in adult hepatocytes (P < 0.005). GLUT-1 protein paralleled its message. GLUT-2 mRNA was more abundant in adult than in fetal hepatocytes (P < 0.05), and abundance did not change during culture, but GLUT-2 protein was discordantly regulated. There was more GLUT-2 protein in fetal hepatocytes at 45 h (P < 0.025). An Eadie-Hofstee plot of 3-O-methylglucose transport appeared to have two linear components. One component was presumed to be GLUT-1 [variable Michaelis constant (Km) approximating 6-8 mM, maximal uptake rate (Vmax) for fetal vs. adult hepatocytes 106 vs. 35 nmol.min-1.mg protein-1], and a second was presumed to be GLUT-2 (Km of 23 mM, Vmax for fetal vs. adult hepatocytes 198 vs. 92 nmol.min-1.mg protein-1). Early phosphorylation of 2-deoxyglucose was greater in fetal than in adult hepatocytes, but transport was always greater than phosphorylation. Increased expression of both GLUT-1 and GLUT-2 by fetal hepatocytes permits greater glucose uptake and positions the fetal rat hepatocyte for efficient glycogenesis at low plasma glucose concentration.
Lipid abnormalities in tissues of the KKAy mouse: effects of pioglitazone on malonyl-CoA and diacylglycerol
Authors: Saha A. K. , Kurowski T. G. , Colca J. R. , Ruderman N. B.
Published: December 2017

Insulin resistance is present in liver and muscle of subjects with type 2 diabetes and obesity. Recent studies suggest that such insulin resistance could be related to abnormalities in lipid-mediated signal transduction; however, the nature of these abnormalities is unclear. To examine this question further, tissue levels of diacylglycerol (DAG), malonyl-CoA, and triglyceride (TG) were determined in liver and soleus muscle of obese insulin-resistant KKAy mice and lean C57 BL control mice. In addition, the effects of treatment with pioglitazone, an antidiabetic agent that acts by increasing insulin sensitivity in muscle, liver, and other tissues, were assessed. The KKAy mice were hyperglycemic (407 vs. 138 mg/dl), hypertriglyceridemic (337 vs. 109 mg/dl), hyperinsulinemic (631 vs. 15 mU/ml), and weighed more (42 vs. 35 g) than the control mice. They also had 1.5- to 2.0-fold higher levels of malonyl-CoA in both liver and muscle, higher DAG (twofold) and TG (1.3-fold) levels in muscle, and higher TG (threefold), but not DAG, levels. Treatment of the KKAy mice with pioglitazone for 4 days decreased plasma glucose, TGs, and insulin by approximately 50% and restored hepatic and muscle malonyl-CoA levels to control values. In contrast, pioglitazone increased hepatic and muscle DAG levels two- or threefold. It has no effect on muscle or hepatic TG content, and it slightly increased hepatic TGs in the control group. The results indicate that abnormalities in tissue lipids occur in both liver and muscle of the KKAy mouse and that they are differentially altered when insulin sensitivity is enhanced by treatment with pioglitazone.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of cell function by the cellular hydration state
Authors: Haussinger D. , Lang F. , Gerok W.
Published: December 2017

Cellular hydration can change within minutes under the influence of hormones, nutrients, and oxidative stress. Such short-term modulation of cell volume within a narrow range acts per se as a potent signal which modifies cellular metabolism and gene expression. It appears that cell swelling and cell shrinkage lead to certain opposite patterns of cellular metabolic function. Apparently, hormones and amino acids can trigger those patterns simply by altering cell volume. Thus alterations of cellular hydration may represent another important mechanism for metabolic control and act as another second or third messenger linking cell function to hormonal and environmental alterations.
Postprandial modulation of lipoprotein lipase in rats with insulin resistance
Authors: Boivin A. , Montplaisir I. , Deshaies Y.
Published: December 2017

The purpose of this study was to determine whether the postprandial modulation of lipoprotein lipase (LPL) activity was altered in rats with resistance of glucose metabolism to insulin action induced by a high-fat diet. Relationships between serum insulin and tissue LPL activity were established in rats chronically fed a high-carbohydrate or high-fat diet, and the effects of fasting and intake of meals of habitual and alternate composition were contrasted. The feeding paradigm did not result in the development of obesity. Global resistance of glucose metabolism to insulin brought about by chronic high-fat feeding was confirmed by an intravenous glucose tolerance test. Fasting serum glucose and insulin concentrations were similar in both cohorts, as was LPL activity in retroperitoneal and inguinal white adipose tissues (WAT), the heart, and soleus. A high-carbohydrate meal brought about higher postprandial insulinemia in the cohort chronically fed the high-fat diet. This was associated with larger changes in LPL activity, that is, an increase in inguinal WAT and in brown adipose tissue and a decrease in soleus, red vastus lateralis, and the heart. Thus the established postprandial modulation of LPL, presumably by insulin, was potentiated in the presence of hyperinsulinemia induced by chronic high-fat feeding despite the concomitant impairment of glucose metabolism.
Interleukin-1 receptor antagonist prevents sepsis-induced inhibition of protein synthesis
Authors: Cooney R. , Owens E. , Jurasinski C. , Gray K. , Vannice J. , Vary T.
Published: December 2017

To understand the role of interleukin-1 (IL-1) as a mediator of the sepsis-induced skeletal muscle catabolism, we investigated the effects of a specific IL-1 receptor antagonist (IL-1ra) on skeletal muscle protein metabolism in a rodent model of chronic abdominal sepsis. A constant infusion of IL-1ra (2 mg.kg-1.h-1) or saline was begun immediately after the induction of sepsis and continued for 5 days. The effect of IL-1ra on protein metabolism was examined in individual muscles (gastrocnemius, soleus, heart) containing different fiber types. Infusion of IL-1ra in control animals did not alter protein metabolism in any of the muscles examined. Muscle weight, protein content, and the rate of protein synthesis in gastrocnemius were reduced by sepsis, whereas none of these parameters were affected in soleus or heart. Infusion of IL-1ra prevented the sepsis-induced loss of muscle protein and inhibition of protein synthesis in gastrocnemius but was without effect in soleus or heart. IL-1ra infusion restored translational efficiency in the gastrocnemius of septic rats and was without effect on the RNA content. These results provide evidence for a role of IL-1 as a mediator of the sepsis-induced abnormalities in skeletal muscle protein metabolism.
Perinatal ontogeny of porcine nuclear thyroid hormone receptors and its modulation by thyroid status
Authors: Duchamp C. , Burton K. A. , Herpin P. , Dauncey M. J.
Published: December 2017

Induction of nuclear thyroid hormone receptors (TRs) represents a key point in the control of growth, development, differentiation, and metabolism of most tissues. The influence of thyroid status on the ontogeny of hepatic and skeletal muscle TRs has been investigated in perinatal pigs. Plasma concentrations of total and free 3,5,3'-triiodothyronine (T3) increased markedly from 80 days of fetal life (80 f) to 2 days of postnatal life. Test piglets obtained from sows fed a high glucosinolate rapeseed diet had lower T3 and thyroxine levels than controls at 110 f and showed a higher postnatal surge in T3. Maximal T3 binding capacity (Bmax, pmol T3/mg DNA, means +/- SE) in liver increased from 0.07 +/- 0.01 at 80 f to 0.37 +/- 0.02 at birth and then plateaued. In longissimus dorsi muscle, Bmax values were much higher than in liver and increased from 0.90 +/- 0.02 at 80 f to 1.37 +/- 0.13 at birth and then declined to 1.09 +/- 0.11 at 2 days of age. Long-term fetal hypothyroidism affected the ontogenic profile of both liver and muscle receptors but in opposite directions; Bmax values were reduced in liver but increased in muscle. Postnatally, lower muscle Bmax values occurred in parallel with transient higher levels of circulating T3. Apparent binding affinities were slightly different in liver and muscle during fetal life, and there was an effect of age in muscle. In conclusion, as far as the receptor is concerned, fetal muscle can potentially respond to thyroid hormones much earlier in development than the liver.(ABSTRACT TRUNCATED AT 250 WORDS)
Differential effects of GLUT-1 or GLUT-4 overexpression on insulin responsiveness in transgenic mice
Authors: Marshall B. A. , Mueckler M. M.
Published: December 2017

The effect of glucose transporter expression on insulin-stimulated whole body glucose disposal was examined in transgenic mice overexpressing GLUT-1 or GLUT-4. Transgenic mice and their control littermates were subjected to a euglycemic hyperinsulinemic clamp under pentobarbital sodium anesthesia using an insulin infusion rate of 20 mU.kg-1.min-1 and a variable glucose infusion rate (GIR). Fasted mice overexpressing GLUT-1 in skeletal muscle exhibited a GIR that was only 54% that of controls (19.3 +/- 1.8 vs. 36.0 +/- 3.9 mg.kg-1.min-1) when blood glucose was clamped at euglycemic values. In contrast, fasted mice overexpressing GLUT-4 in fat and muscle exhibited a GIR that was 40% higher than controls (53.9 +/- 2.3 vs. 39.1 +/- 2.5 mg.kg-1.min-1). At the end of the clamp, beta-hydroxybutyrate levels were 10-fold higher in the GLUT-1 transgenic mice relative to nontransgenic littermates (2.0 +/- 0.6 vs. 0.2 +/- 0.1 mM) but did not differ between the GLUT-4 transgenic mice and their control littermates (0.3 +/- 0.1 vs. 0.3 +/- 0.1 mM). These data demonstrate that the level of expression of a glucose transporter in muscle and fat can have marked effects on whole body glucose homeostasis and fuel metabolism. Insulin responsiveness was enhanced by overexpression of GLUT-4. Strikingly, however, overexpression of GLUT-1 in muscle induced a profound reduction in insulin-stimulated whole body glucose disposal.(ABSTRACT TRUNCATED AT 250 WORDS)
A new method for the estimation of the components of energy expenditure in patients with major trauma
Authors: Franch-Arcas G. , Plank L. D. , Monk D. N. , Gupta R. , Maher K. , Gillanders L. , Hill G. L.
Published: December 2017

The management of critically ill patients would be better understood if the total energy expenditure (TEE) and its components are known. To quantify the different components of energy expenditure in patients with major trauma, we used a technique combining measurements of body composition and oxygen consumption. We determined changes in body weight, total body water, total body protein, total body potassium, total body fat, and bone mineral content every 5 days over a 10-day period in a group of nine multiply injured patients. Resting energy expenditure was measured by indirect calorimetry (REEm), and a predicted value was obtained from total body potassium (REEp). TEE was assessed by adding the total calorie intake to the changes in body energy stores, and the activity energy expenditure (AEE) was calculated by subtracting REEm from TEE. Mean daily values for REEm, REEp, TEE, and AEE were 2,236 +/- 140, 1,683 +/- 82, 3,029 +/- 276, and 793 +/- 213 kcal/day, respectively, over the 10-day study period. Although not statistically significant, the mean AEE was four times smaller for the first 5 days of study than for the second 5 days (298 +/- 400 vs. 1,254 +/- 588 kcal/day). The technique of combining indirect calorimetry and body composition measurements offers a new approach to evaluate energy expenditure and a new way to study metabolic disorders and therapeutic strategies in critically ill patients.
Muscle growth in response to mechanical stimuli
Authors: Goldspink D. F. , Cox V. M. , Smith S. K. , Eaves L. A. , Osbaldeston N. J. , Lee D. M. , Mantle D.
Published: December 2017

The relative merits of the separate and combined uses of stretch and electrical stimulation at 10 Hz in influencing the rates of protein synthesis in vivo, proteolysis, and the growth of the extensor digitorum longus muscle have been investigated after 3 days in the rabbit. Continuous electrical stimulation failed to change muscle protein turnover or growth. Static stretch caused significant adaptive growth, with increases in c-fos, c-jun, and insulin-like growth factor I (IGF-I; 12-fold) mRNA levels, and protein (19%), RNA (128%), and DNA (45%) contents. Both the fractional (138%) and total (191%) rates of protein synthesis increased with stretch, correlating with increased ribosomal capacities. Combining stretch and electrical stimulation increased the mRNA concentration of IGF-I (40-fold). The adaptive growth was greater (35%), with massive increases in the nucleic acids (185 and 300%), ribosomal capacities (230%), and the rates of protein synthesis (345 and 450%). Large increases (i.e., 200-400%) in cathepsins B and L and dipeptidyl aminopeptidase I activities during stretch, with or without stimulation, suggest a role for these enzymes in tissue remodeling during muscle hypertrophy.
Inhibition of insulin-mediated glucose uptake in rat hindlimb by an alpha-adrenergic vascular effect
Authors: Rattigan S. , Dora K. A. , Colquhoun E. Q. , Clark M. G.
Published: December 2017

The vasoconstrictor norepinephrine, at high doses, inhibits oxygen uptake (VO2) in the perfused hindlimb, possibly by opening vascular shunts and reducing nutrient access. Thus, in the present study, the effect of norepinephrine on insulin-mediated glucose uptake (IMGU) was assessed. Rat hindlimbs were perfused at constant flow with medium containing 8.3 mM glucose and a tracer amount of 2-deoxy-D-[1-3H]glucose (2-DG) with and without 15 nM insulin, 10 microM norepinephrine (NE), and combinations of the adrenergic blockers propranolol (Prop) and prazosin. Perfusions were also conducted at a lower dose of 1 microM NE where VO2 is stimulated. NE (10 microM) inhibited IMGU > 80%, and this inhibition, when measured by 2-DG uptake, was most pronounced in muscles rich in white fibers. The inhibitory effect of NE on IMGU comprised a beta-adrenergic component also partly evident at lower concentrations of NE (i.e., 1 microM) and an alpha-adrenergic component only evident at 10 microM NE. In contrast to the results for the hindlimb, 10 microM NE plus Prop (alpha-adrenergic combination) had no significant effect on insulin-mediated 2-DG uptake by isolated incubated soleus or extensor digitorum longus muscles. It is concluded that NE, at doses likely to occur at sympathetic vasoconstrictor synapses in muscle, impairs IMGU by a vascular effect to cause shunting and reduce access.
Metabolic alterations associated with the antidiabetic effect of beta 3-adrenergic receptor agonists in obese mice
Authors: Arbeeny C. M. , Meyers D. S. , Hillyer D. E. , Bergquist K. E.
Published: December 2017

Treatment of obese (ob/ob) mice with the beta 3-adrenergic receptor (beta 3-AR) agonist BRL-35135 (1 mg.kg body wt-1.day-1 for 20 days) normalized plasma glucose levels and significantly decreased plasma insulin and nonesterified fatty acid levels. The time frame for the hypoglycemic effect, which reached a maximum after 10 days of treatment, paralleled an increase in brown adipose tissue DNA and protein content. The basal level of mRNA for the beta 3-AR and mitochondrial uncoupling protein was found to be markedly decreased in the ob/ob animals relative to the lean group. Chronic treatment of ob/ob mice for 20 days resulted in a twofold increase in beta 3-AR mRNA and a fivefold increase in uncoupling protein mRNA in brown adipose tissue relative to the placebo group. These findings indicate that chronic treatment of ob/ob animals with a beta 3-AR agonist results in proliferation of brown adipose tissue, with an upregulation of the beta 3-AR, which is associated with a decrease in plasma glucose, insulin, and nonesterified fatty acid levels.
Evidence for regulated coupling of A1 adenosine receptors by phosphorylation in Zucker rats
Authors: Berkich D. A. , Luthin D. R. , Woodard R. L. , Vannucci S. J. , Linden J. , LaNoue K. F.
Published: December 2017

Studies were designed to find the molecular basis for previous observations that lipolysis is less active and A1 adenosine receptor signaling is more active in adipocytes from obese than from lean Zucker rats. With quantitative immunoblot procedures for detection, Gi alpha 1 and Gs alpha 45 levels were found anomalously low in obese compared with lean membranes (50 and 30%, respectively), but other G alpha subunit levels were normal. However, the sensitivity of the receptor-Gi protein to GTP was about 5- to 10-fold higher in obese compared with lean membranes when assessed from 1) the ability of GTP to inhibit forskolin-stimulated adenylyl cyclase in the presence of an adenosine receptor agonist and 2) the ability of a nonhydrolyzable guanine nucleotide analogue to alter A1 adenosine receptor agonist binding. Alkaline phosphatase treatment of isolated adipocyte membranes from obese but not lean animals decreased guanine nucleotide sensitivity of agonist binding. Surprisingly, solubilized adipocyte A1 adenosine receptors from all animals exhibited the same high sensitivity to guanine nucleotides as that of intact obese membranes, and this high sensitivity could be decreased 20-fold by treatment with alkaline phosphatase. These data suggest that protein phosphorylation may regulate coupling of the A1 adenosine receptor in rat adipocyte membranes.