bluuebunny/crossref_metadata_2025 huggingface dataset:
Vasopressin uptake by hypothalamopituitary axis and pineal gland in guinea pigs
Authors: Zlokovic B. V. , Hyman S. , McComb J. G. , Tang G. , Rezai A. R. , Weiss M. H.
Published: December 2017

The uptake of circulating arginine vasopressin (AVP) by the pituitary gland (anterior lobe), pineal gland, and hypothalamus (ventromedial part) was investigated in an isolated in situ perused brain of anesthetized guinea pig. Kinetic experiments revealed saturable AVP uptake in all studied regions with Km values of 0.79, 0.19, and 0.76 microM and maximum velocity values of 22, 2.1, and 1.6 pmol.min-1.g-1 for the pituitary gland, pineal gland, and hypothalamus, respectively. The nonsaturable components (diffusion constants) were not significantly different from zero. Peptide fragments, L-phenylalanine, and Bestatin (an aminopeptidase inhibitor) did not interfere with AVP uptake. However, uptake of AVP was strongly inhibited in the presence of the V1 antagonist [1-(beta-mercapto-beta-beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine]AVP at 2.7 microM, and regional Ki values, assuming that the observed inhibitions were purely competitive, ranged between 0.32 and 2.23 microM. The V2 agonist 1-desamino-8-D-AVP at 2.7 microM produced only a weak inhibition of AVP uptake, and regional Ki values ranged between 9.56 and 21.3 microM. It is concluded that specific uptake mechanisms in the hypothalamopituitary axis and pineal gland are sensitive enough to detect blood-borne AVP under the physiological hormonal state. It is suggested that AVP binding in situ is primarily related to V1 receptors, which may be involved in mediating the central effects of this circulating peptide.
Cortical bone maturation in young hypophysectomized rats
Authors: Martinez D. A. , Vailas A. C. , Grindeland R. E.
Published: December 2017

Cortical bone growth and maturation were studied in young growing rats (62 days) that were hypophysectomized. After 10 days, femur bones were obtained for morphological and biochemical analysis of the middiaphysis. Hypophysectomy significantly decreased (42%) normal body growth demonstrated by reductions in body weight. Bone morphology data indicate that hypophysectomy significantly inhibits the circumferential growth of the middiaphysis as well as region-specific thickness compared with the control group. The effects of hypophysectomy on cortical bone mineral composition and density were significantly greater in the hypophysectomized rats. Bone density and total mineral density were significantly greater, 17% and 24%, respectively, in the hypophysectomized group compared with control animals. Bone collagen maturation, measured by the amount of the mature collagen cross-link hydroxylysylpyridinoline, in the hypophysectomized animals was significantly greater (130%). Also, collagen concentration and content per unit volume were significantly greater, 24% and 70%, respectively, in the hypophysectomized group. Our findings suggest that "normal" growth and maturation of cortical bone in rapidly growing animals is dependent upon the modulation of pituitary hormones. These data also suggest that an increased rate of mature collagen cross-link formation may be modified by the turnover rate of collagen and other matrix constituents. In addition, hormonal mechanisms associated with a suppression of bone growth may enhance the probability of mature collagen cross-link formation.
Differential effect of hyperglycemia and hyperinsulinemia on pathways of hepatic glycogen repletion
Authors: Shulman G. I. , DeFronzo R. A. , Rossetti L.
Published: December 2017

To delineate the roles of hyperglycemia and insulin on the direct vs. indirect pathways of liver glycogen synthesis, we performed euglycemic (group I; n = 8), hyperglycemic (group II; n = 9), and euglycemic pharmacological hyperinsulinemic clamp studies (120 min) with an infusion of [1-13C]glucose in chronically catheterized conscious rats after a 24-h fast. Portal vein plasma glucose concentrations and portal vein plasma insulin concentrations, respectively, obtained at the end of the study in groups I-III were as follows: group I 110 +/- 4 mg/dl, 29 +/- 7 ng/ml; group II 219 +/- 7 mg/dl, 24 +/- 7 ng/ml; and group III 112 +/- 9 mg/dl, 174 +/- 25 ng/ml. Mean liver glycogen concentrations at the end of the three studies were 0.68 +/- 0.07, 1.22 +/- 0.08 (P less than 0.001 compared with groups I and III), and 0.60 +/- 0.17 g/100 g wet wt liver in groups I-III respectively, which yielded hepatic glycogen synthetic rates of 0.16 +/- 0.03, 0.41 +/- 0.04 (P less than 0.001 compared with groups I and III), and 0.13 +/- 0.08 mumol glucosyl U.g liver-1.min-1 in groups I-III, respectively. From the enrichments of 13C in the C-1 and C-6 positions of the glucosyl unit in glycogen compared with the enrichment in the C-1 position in portal vein glucose as determined by 13C- and 1H-NMR, the amount of glycogen synthesized by the direct pathway was calculated to be 18 +/- 2, 41 +/- 3 (P less than 0.0001 compared with groups I and III), and 17 +/- 3% in groups I-III, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
ANG II blocks potassium currents in zona glomerulosa cells from rat, bovine, and human adrenals
Authors: Brauneis U. , Vassilev P. M. , Quinn S. J. , Williams G. H. , Tillotson D. L.
Published: December 2017

Angiotensin II (ANG II) is a principal secretagogue of adrenal zona glomerulosa (ZG) cells. The transduction process includes a depolarization of the plasma membrane and the activation of calcium influx. The ANG II-induced depolarization is associated with an increase in total membrane resistance. To directly address the mechanism underlying these observations, we examined the effect of ANG II on K+ currents of rat, bovine, and human ZG cells, using whole cell patch clamp. Although some differences were seen in the characteristics of K+ currents between species, ANG II consistently blocked outward currents in ZG cells [rat: 47.1 +/- 4.5% (SE), n = 17; bovine: 38.6 +/- 3.3%, n = 21; and human: 13-63%, n = 3]. With the use of the cell-attached mode, single-channel recordings in bovine ZG cells demonstrated K+ channels that were reversibly blocked when ANG II was added to the bath solution. This indicates that the block of K+ channels by ANG II involves a diffusible intracellular messenger rather than a direct receptor-channel interaction. The decreased conductance of K+ can account for the ANG II-induced membrane depolarization.
Differential effects of omega-3 fish oils on protein kinase activities in vitro
Authors: Speizer L. A. , Watson M. J. , Brunton L. L.
Published: December 2017

We studied the in vitro effects of omega-3 fish oils and other fatty acids on the activity of crude protein kinase C from S49 lymphoma cells, on partially purified enzyme from rat cerebrum, on homogeneous protein kinase C from bovine brain, and, for comparison, on type I adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. In the absence of exogenous phospholipid, the fish oils cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and acid (DCHA) enhance the catalytic cis-4,7,10,13,16,19-docosahexaenoic activity of protein kinase C and support the binding of [3H]phorbol 12,13-dibutyrate, both to approximately 50% of the level supported by phosphatidylserine. In the presence of phosphatidylserine, the omega-3 fatty acids reduce catalytic activity and [3H]phorbol 12,13-dibutyrate binding by about one-half. The effects of the omega-3 fatty acids on enzyme activity suggest that fish oils act as partial agonists competitively with phosphatidylserine. EPA, DCHA, and arachidonate (but not a variety of saturated fatty acids) inhibit the cAMP-dependent protein kinase. Thus dietary fish oils and cellular fatty acids mobilized by the action of phospholipase A2 may differentially modulate the activities of protein kinase C and cAMP-dependent protein kinase. These data suggest means by which unsaturated fatty acids mobilized within cells may act as second messengers.
Nuclear calcium gradients in cultured rat hepatocytes
Authors: Waybill M. M. , Yelamarty R. V. , Zhang Y. L. , Scaduto R. C. , LaNoue K. F. , Hsu C. J. , Smith B. C. , Tillotson D. L. , Yu F. T. , Cheung J. Y.
Published: December 2017

Ca2+ concentrations ([Ca2+]) in cytosol and nucleus in fura-2-loaded cultured rat hepatocytes were determined by three-dimensional (3-D) optical-sectioning microscopy. After determining the empirical 3-D point spread function of the fluorescence microscope-coupled digital video imaging system, contaminating light arising from optical planes above and below the plane of interest was removed by deconvolution using the nearest-neighboring approach (NNA) algorithm. Although deconvolution resulted in substantial improvement in accuracy of fluorescence intensity determinations in single-wavelength excitation images as well as sharper delineation of boundaries between cellular compartments, the complicated mathematical process did not significantly enhance the precision of [Ca2+] values derived from ratiometric (ratio of dual-wavelength excitation) images. In resting hepatocytes, cytosolic Ca2+ (210 +/- 15 nM) was 1.6- to 2-fold higher than nuclear Ca2+ (128 +/- 12 nM). This difference in Ca2+ between the two compartments was detected both in raw ratiometric images and in those processed with NNA algorithm. Addition of arginine vasopressin or epidermal growth factor resulted in significant increases (2- to 3-fold) in both cytosolic and nuclear Ca2+; however, the nuclear-to-cytosolic Ca2+ gradient was preserved in hepatocytes stimulated with mitogens. We conclude that the hepatocyte nuclear membrane contains Ca2+ permeability barriers and Ca2+ transport mechanisms that may be hormonally sensitive. We postulate that the increase in nuclear Ca2+ may be important in regulation of cell proliferation induced by mitogens, possibly by activating Ca(2+)-dependent endonucleases, nuclear calmodulin, or nuclear protein kinase C.
On the determination of turnover in vivo with tracers
Authors: Katz J.
Published: December 2017

Theoretical and practical aspects of the application of tracer methods for the measurement of turnover of blood-borne compounds are discussed, with special regard to lactate. The validity of the application of the tracer into the aortic arch and sampling from the right atrium (A-V), the administration of tracer into the vena cava and sampling from the aorta (V-A), and sampling to the determination of turnover are examined, using numerical examples. It is shown that the difference between specific activity in arterial and mixed venous blood depends mainly on the cardiac output, the ratio of tracee turnover to the mass of circulating tracee, and the sites of production and utilization of the tracee. Conditions are shown under which the A-V and V-A modes overestimate or underestimate the true rate of turnover. In theory, the A-V mode provides an exact estimate of turnover when the mean specific activity of the tracee in the whole body equals the specific activity of mixed venous blood in the right heart. It is shown that, for compounds with a high turnover rate, the underestimate in the A-V mode is small, and the mode provides a close approximation of true turnover. The underestimate in the V-A mode at high turnover rates is extensive. Experimental evidence indicates that, in several animal species, the specific activity of lactate and several amino acids in several organs and tissues nearly equals that in the venous blood, with the A-V mode providing a close approximation of the true turnover for these compounds.(ABSTRACT TRUNCATED AT 250 WORDS)
Fasting and lactate unmask insulin responsiveness in the isolated working rat heart
Authors: Russell R. R. , Nguyen V. T. , Mrus J. M. , Taegtmeyer H.
Published: December 2017

We have previously reported that the nutritional state in vivo results in differential insulin responses by the perfused heart in vitro. To further assess the effects of insulin on glucose uptake at physiological work loads, hearts from fed and fasted (16-20 h) rats were perfused with buffer containing 2-[18F]fluoro-2-deoxy-D-glucose (2-FDG) and glucose (10 mM) alone or plus lactate (10 mM) as a competing substrate, with insulin (10 mU/ml) added after a control period. When glucose was the only substrate, the addition of insulin decreased the fractional rate of 2-FDG uptake in hearts from either fed or fasted rats. The effect of insulin on increasing myocardial 2-FDG uptake was immediate and sustained only in hearts from fasted rats in the presence of lactate, despite no change in cardiac work. At the same time, the increase in 2-FDG uptake and phosphorylation was associated with an increase in the tissue content of glycogen in hearts from fasted rats. We conclude that lactate unmasks insulin sensitivity in heart muscle at physiological work loads but that this unmasking of insulin-mediated glucose uptake is dependent on the nutritional state of the animal. The glucose up as a result of insulin stimulation is preferentially utilized for glycogen repletion and does not enter the glycolytic pathway. This observation also suggests that myocardial glycogen synthesis in vitro is affected by the nutritional state in vivo and that lactate provides a substrate for oxidative phosphorylation while glucose is preferentially utilized for glycogen synthesis.
Pretranslational regulation of two cardiac glucose transporters in rats exposed to hypobaric hypoxia
Authors: Sivitz W. I. , Lund D. D. , Yorek B. , Grover-McKay M. , Schmid P. G.
Published: December 2017

To investigate the mechanism by which cardiac glucose utilization increases during hypoxia and increased work load, we studied the effect of 2 and 14 days of hypobaric hypoxia on the expression of two subtypes of the facilitative D-glucose transporter, the GLUT-4 or "insulin-regulatable" isoform and the GLUT-1 isoform thought to mediate basal transport. Rats lose weight when exposed to hypobaric hypoxia, so fasting controls were used in the 2-day studies and pair-fed controls in the 14-day experiments. Hypobaric hypoxia (PO2 69 mmHg) resulted in right ventricular (RV), but not left ventricular (LV), hypertrophy. RV and LV GLUT-1 mRNA levels increased 2- to 3-fold after 2 days and 1.5- to 2-fold after 14 days of hypobaric hypoxia compared with both fasted rats and normal controls. RV GLUT-1 protein increased approximately 3-fold and LV GLUT-1 protein increased 1.5-fold after 14 days of hypobaric hypoxia vs. both pair-fed and normal controls. RV GLUT-4 mRNA decreased to 26% and RV GLUT-4 protein decreased to 54% of normal control levels as a result of 2 days of hypobaric hypoxia. RV GLUT-4 mRNA decreased to 64% of normal control levels with no change in RV GLUT-4 protein as a result of 2 days of fasting. We conclude that hypobaric hypoxia increases cardiac GLUT-1 expression at the pretranslational level in both ventricles. The greater increase in GLUT-1 protein on the right suggests an additive effect of pressure overload. GLUT-4 expression is reduced early in the development of RV hypertrophy.
Sulfate conjugates of iodothyronines in developing sheep: effect of fetal hypothyroidism
Authors: Wu S. Y. , Polk D. H. , Huang W. S. , Reviczky A. , Wang K. , Fisher D. A.
Published: December 2017

We recently showed that thyroxine sulfate (T4S) and 3,3',5-triiodothyronine sulfate (T3S) were major thyroid hormone metabolites in ovine fetuses and neonates. To further characterize the sulfation pathway in ovine fetuses, we measured 3,3',5'-triiodothyronine (rT3S) in serum and other body fluids in samples obtained from fetal (n = 23, 94-145 days of gestational age, term = 150 days), newborn (n = 6), and adult (n = 6) sheep. In addition, T3S, T4S, and rT3S levels were measured in tissue fluids and serum samples obtained from ovine fetuses 13 days after total thyroidectomy (Tx) conducted at gestational age of 110-113 days (n = 5). Sham-operated twin fetuses served as controls (n = 5). The relative order of mean rT3S concentration for various tissue fluids in fetuses were meconium > bile > serum > allantoic fluid > urine or amniotic fluid. Peak mean tissue fluid levels generally occurred at 110-130 days gestation. In hypothyroid fetuses, significant decreases in the mean serum concentrations of T4S and rT3S, but not T3S, were noted. The mean rT3S level also was decreased significantly in allantoic fluid, bile, and meconium, whereas T4S and T3S levels were reduced only in bile of the Tx fetuses. These data demonstrate that sulfation is a major pathway in thyroid hormone metabolism in both euthyroid and hypothyroid ovine fetuses.
Okadaic acid, insulin, and denervation effects on glucose and amino acid transport and glycogen synthesis in muscle
Authors: Robinson K. A. , Boggs K. P. , Buse M. G.
Published: December 2017

Effects of okadaic acid (OKA) and calyculin A, cell-permeating specific inhibitors of phosphoprotein phosphatases-1 and -2A, were studied in isolated rat hemidiaphragms. OKA stimulated glucose transport (half-maximum = approximately 0.1 microM; maximum = approximately 1 microM) but was less effective than 6 nM insulin. Insulin and OKA effects were not additive. OKA diminished or abolished glucose transport-stimulation by insulin. System A amino acid transport was also stimulated by OKA, insulin was more effective, and preexposure to OKA inhibited insulin stimulation. Calyculin A affected both transport systems similarly to OKA. OKA did not affect basal glycogen synthesis but abolished its stimulation by insulin. Denervated muscles develop post-receptor insulin resistance. Glucose transport and glycogen synthesis were essentially unresponsive to insulin 3 days postdenervation; however, glucose transport was stimulated by OKA similarly to controls. OKA did not affect glycogen synthesis in denervated muscle except for abolishing a small insulin effect. The data suggest similar acute regulation of glucose and system A amino acid transport in muscle. Enhanced Ser/Thr phosphorylation of unidentified protein(s) stimulates both processes but inhibits their full stimulation by insulin. Postdenervation insulin resistance likely reflects impaired signal transduction.
Regulation of gluconeogenesis during rest and exercise in the depancreatized dog
Authors: Wasserman D. H. , Johnson J. L. , Bupp J. L. , Lacy D. B. , Bracy D. P.
Published: December 2017

To assess the mechanism of the accelerated gluconeogenesis in the insulin-deficient state, chronically catheterized (carotid artery, portal vein, hepatic vein, vena cava) normal (C; n = 9) and depancreatized (PX; n = 7) dogs were studied during rest (40 min) and moderate exercise (150 min). Tracers ([14C]alanine, [3H]glucose) and dye were infused to measure determinants of gluconeogenesis in the gut and liver. Arterial levels, net gut output, hepatic load, and net hepatic uptake of alanine were similar in C and PX at rest. During exercise, alanine levels fell in C but rose approximately 100% in PX. Exercise did not affect gut output or liver uptake of alanine in C but increased these variables by approximately 50 and 100% in PX due to an increase in hepatic alanine load. Arterial lactate was similar at rest in C and PX but rose fourfold more in PX with exercise. Net gut lactate output was fivefold greater in PX during rest and exercise. Net hepatic lactate uptake was present in PX at rest, whereas net output was evident in C. In response to exercise, hepatic lactate uptake was increased further in PX due to a rise in hepatic lactate load. Net hepatic lactate uptake was not evident until the end of exercise in C. Net hepatic glycerol uptake was elevated at rest in PX and during the initial 60 min of exercise due to an elevated hepatic load. In contrast to the high rates of gut lactate and alanine output in PX, gut glycerol output was not present. Gluconeogenesis from lactate and alanine was 5- to 10-fold higher in PX than C during rest and exercise. At rest, this resulted, in part, from a twofold greater intrahepatic gluconeogenic efficiency. During exercise, the greater conversion occurred even though efficiency was not consistently greater. In summary, gluconeogenesis from alanine, lactate, and glycerol in the insulin-deficient diabetic state 1) is exaggerated at rest, due to an increased capacity for hepatic lactate extraction, increased hepatic precursor loads, and a greater gluconeogenic efficiency; 2) is accelerated further by exercise due to added increments in hepatic precursor loads; and 3) is exaggerated partly because of a greater net gut alanine and lactate output.
Increased clearance of 1,25(OH)2D3 and tissue-specific responsiveness to 1,25(OH)2D3 in diabetic rats
Authors: Verhaeghe J. , Suiker A. M. , Van Bree R. , Van Herck E. , Jans I. , Visser W. J. , Thomasset M. , Allewaert K. , Bouillon R.
Published: December 2017

The kinetics of 1,25-dihydroxyvitamin D3 [1,25(OH)2-D3] and the in vivo response to 1,25(OH)2D3 (7.5, 15, and 30 ng/100 g body wt), infused or injected subcutaneously for 12-14 days, were studied in male spontaneously diabetic and control BB rats. In control rats, increasing doses of 1,25(OH)2D3 produced parallel increases in plasma 1,25(OH)2D3 and calcium, urinary calcium, duodenal CaBP9K, and renal CaBP28K. 1,25-(OH)2D3 at 30 ng/100 g markedly raised plasma osteocalcin and osteoblast/osteoid surfaces in the tibial metaphysis, but inhibited bone mineralization rate. In diabetic rats, plasma 1,25-(OH)2D3 concentrations were decreased, and the rise of plasma 1,25(OH)2D3 during 1,25(OH)2D3 infusion was blunted, but the free 1,25(OH)2D3 index remained normal or above normal. Diabetic rats had an increased metabolic clearance rate of 1,25-(OH)2D3 (0.38 +/- 0.015 vs. 0.24 +/- 0.007 ml.min-1.kg-1), with no further increase in 1,25(OH)2D3-infused diabetic rats; their relative production rate of 1,25(OH)2D3 was unchanged. The responses of plasma and urinary calcium, duodenal CaBP9K, and renal CaBP28K to infused 1,25(OH)2D3 were normal, as was duodenal calcium absorption in 1,25(OH)2D3-injected diabetic rats. However, the virtual absence of osteoblasts/osteoid in trabecular bone was unaltered in diabetic rats infused with 30 ng/100 g 1,25(OH)2D3, with only minimal increase of their low plasma osteocalcin levels. 1,25(OH)2D3 treatment therefore cannot be expected to reverse diabetic osteopenia.
Dietary arginine uptake by the splanchnic region in adult humans
Authors: Castillo L. , Chapman T. E. , Yu Y. M. , Ajami A. , Burke J. F. , Young V. R.
Published: December 2017

To determine the uptake of dietary arginine and leucine by the splanchnic region, two experiments were carried out, each involving four healthy young adult men who received a diet supplying 1 g protein.kg-1.day-1 for 7 and 10 days before conducting a primed constant tracer infusion protocol. In study 1, subjects received for 8 h (3-h fast; 5-h fed state, achieved by a constant intragastric infusion of the diet formula) L-[5,5-2H2; guanidino-15N2]arginine ([M4]Arg), L-[guanidino-13C]arginine ([13C]Arg), and L-[5,5,5-2H3]leucine ([2H3]Leu) simultaneously by an intragastric infusion on day 7 and a repeat of this protocol on day 10 except with tracer administration given by vein. Plasma arginine fluxes were essentially the same for the two arginine tracers but differed significantly with route of administration. In study 2 the subjects received on day 7 a constant intravenous infusion of [13C]Arg and [2H3]Leu and a simultaneous intragastric infusion of [M4]Arg and [1-13C]leucine. On day 10 the routes of administration of these tracer pairs were reversed. During the fed state in study 1, splanchnic uptake of dietary arginine was 31 +/- 10 and 34 +/- 8%, based on the [13C]Arg and [M4]Arg tracers, respectively, and it was significantly higher (P < 0.01) than for leucine, which was 10 +/- 6%. In study 2, splanchnic uptake of dietary arginine, estimated from a series of tracer-protocol combinations for the fed state, was approximately 38% compared with a lower (P < 0.01) value of approximately 15% for leucine.
Importance of brain IL-1 type II receptors in fever and thermogenesis in the rat
Authors: Luheshi G. , Hopkins S. J. , Lefeuvre R. A. , Dascombe M. J. , Ghiara P. , Rothwell N. J.
Published: December 2017

Interleukin-1 (IL-1) acts centrally to induce fever and thermogenesis in rodents. The central actions of IL-1 alpha and IL-1 beta apparently involve different mechanisms, and the effects of IL-1 beta are not consistent with interaction with a type I (IL-1RI) 80-kDa receptor. In the present study the involvement of the type II IL-1 receptor (IL-1RII) was tested in the rat by examining the effects of central injection of a monoclonal antibody (ALVA-42), which blocks the IL-1RII. Pretreatment of rats with ALVA-42 (6 micrograms icv) inhibited the thermogenic and pyrogenic responses to intracerebroventricular injection of 5 ng (but not 50 ng) of IL-1 beta in conscious rats but did not significantly modify responses to IL-1 alpha. ALVA-42 also failed to modify the responses to peripherally administered IL-1 beta (1 microgram) but significantly attenuated the pyrogenic and thermogenic responses to peripheral (125 micrograms) or central (1 microgram) injection of endotoxin. These data indicate that IL-1RII mediates the central effects of a low dose of IL-1 beta, but not IL-1 alpha, on fever and thermogenesis in the rat. They also imply that responses to endotoxin are due, at least in part, to the activation of IL-1RII by IL-1 beta released within the brain and that effects of peripherally injected IL-1 beta involve different mechanisms, probably associated with IL-1RI.
Increased fetal glucose concentration decreases ovine fetal leucine oxidation independent of insulin
Authors: Liechty E. A. , Boyle D. W. , Moorehead H. , Liu Y. M. , Denne S. C.
Published: December 2017

Fetal leucine oxidation rate is elevated during fasting of the ewe. Euglycemic hyperinsulinemia causes the leucine oxidation rate to decline. However, it is unclear whether this is a direct effect of insulin or is secondary to increased insulin-mediated glucose utilization. To better delineate the mechanism of decreased oxidation, we suppressed fetal insulin secretion by somatostatin infusion. Glucose was infused at a variable rate to achieve glucose concentrations 125 and 150% of basal. Leucine rate of appearance (Ra) was determined by infusion of [15N, 1-13C]leucine. Fraction of leucine appearance oxidized was determined by [1-14C]leucine infusion and determination of fetal 14CO2 excretion. Each fetus was studied during ad libitum maternal feeding and after a 5-day complete maternal fast. Changes were noted in fetal leucine oxidation, which declined from 8.4 +/- 1.2 to 5.0 +/- 0.8 mumol/min in the fed state during glucose infusion. Basal leucine oxidation was elevated during fasting (11 +/- 1.5 mumol/min, P < 0.05) and declined to 8.0 +/- 1.4 mumol/min during glucose infusion (P = 0.056). Leucine carbon Ra was unchanged by fasting and by glucose infusion; leucine nitrogen Ra declined in the fed state only. Leucine oxidation was inversely correlated with glucose concentration (oxidation = 12-0.26 x glucose concentration, r = 0.42, P = 0.004). Leucine oxidation was not correlated with insulin concentration (r = 0.2). Changes in fetal glucose concentration may alter the pattern of utilization of essential amino acids, independent of changes in insulin and insulin-mediated glucose utilization rate.
Effects of insulin on the counterregulatory response to equivalent hypoglycemia in normal females
Authors: Davis S. N. , Cherrington A. D. , Goldstein R. E. , Jacobs J. , Price L.
Published: December 2017

The aim of this study was to determine if insulin could augment the counterregulatory response to equivalent hypoglycemia in normal females similarly to males. Experiments were carried out in nine normal lean overnight-fasted female subjects. Insulin was infused in two separate randomized protocols so that steady-state levels of 794 +/- 62 (low) and 3,620 +/- 476 pM (high) were obtained. Despite an identical plasma glucose level (2.8 +/- 0.1 mM), epinephrine (5.7 +/- 0.9 vs. 3.9 +/- 0.6 nM), norepinephrine (2.7 +/- 0.4 vs. 1.8 +/- 0.3 nM), cortisol (918 +/- 55 vs. 826 nM), and growth hormone (35.8 +/- 3.7 vs. 28.4 +/- 2.7 micrograms/l) were increased (P < 0.05) during high compared with low insulin infusion, respectively. Glucagon and pancreatic polypeptide levels increased significantly but were not different during the two insulin infusions. Hepatic glucose production was increased during the high-compared with low-dose infusions (9.5 +/- 1.1 vs. 5.1 +/- 2.2 mumol.kg-1 x min-1; P < 0.05). Lipolysis, as indicated by the blood glycerol level, increased significantly during high- compared with low-dose insulin infusions (121 +/- 29 vs. 65 +/- 13 microM; P < 0.05). The hormonal and metabolic responses to hypoglycemia were significantly different in females compared with previous results in males.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of fat-carbohydrate interaction in skeletal muscle during intense aerobic cycling
Authors: Dyck D. J. , Putman C. T. , Heigenhauser G. J. , Hultman E. , Spriet L. L.
Published: December 2017

Six male subjects received either a saline (control) or Intralipid infusion during 30 min rest and 15 min cycling at 85% maximal O2 uptake (VO2max) to examine the regulation of fat-carbohydrate interaction (glucose-fatty acid cycle) in skeletal muscle. Muscle biopsies were sampled immediately before and at 3 and 15 min of exercise in both trials. A muscle biopsy was also taken at -30 min rest in the Intralipid trial. Intralipid infusion significantly elevated plasma free fatty acids above control during rest (0.21 +/- 0.04 to 0.94 +/- 0.09 mM) and exercise (5 min: 1.27 +/- 0.15 mM; 15 min: 1.42 +/- 0.13 mM). Muscle glycogen degradation was significantly lower in the Intralipid trial (109.7 +/- 29.3 vs. 194.7 +/- 32.1 mmol/kg dry muscle). Muscle lactate accumulation after 15 min was similar in both trials (control, 60.7 +/- 12.2 and Intralipid, 60.9 +/- 12.4 mmol/kg dry muscle). Muscle citrate increased at rest during Intralipid (0.32 +/- 0.06 to 0.58 +/- 0.06 mmol/kg dry muscle) but was not different between trials at 3 min (control, 0.73 +/- 0.07 and Intralipid, 0.68 +/- 0.06 mmol/kg dry muscle) and 15 min of cycling. Resting acetyl-CoA was unaffected by Intralipid and increased similarly in both trials at 3 min of cycling (control, 59.0 +/- 10.3 and Intralipid, 50.7 +/- 13.6 mumol/kg dry muscle) and remained unchanged at 15 min. Pyruvate dehydrogenase activity increased five- to sevenfold during exercise and was similar in both trials (15 min: control, 2.42 +/- 0.30 and Intralipid, 2.79 +/- 0.41 mmol.min-1 x kg wet wt-1).(ABSTRACT TRUNCATED AT 250 WORDS)
Growth hormone-enhanced acid production and glutamate and glutamine utilization in LLC-PK-F+ cells
Authors: Welbourne T. C. , Fuseler J.
Published: December 2017

Growth hormone (GH), recombinant human (rh) GH, effects on transepithelial proton gradient, acid production, and glutamine (Gln) and glutamate (Glu-) utilization were studied in LLC-PK-F+ cells. Monolayers were incubated in Dulbecco's modified Eagle's media containing either 2 mM Gln or Glu- and 5 mM glucose plus 10% fetal bovine serum for 4 h, after which samples were taken for metabolite and acid-base parameter measurements. Monolayers grown on porous supports in Glu- media responded to 32 nM rhGH by decreasing Glu- net uptake 29% (470 +/- 135 to 336 +/- 98 nmol/4 h, P < 0.05), reflecting elimination of basolateral uptake (189 +/- 80 to 2 +/- 35 nmol/4 h, P < 0.05) as ammonium production decreased. In 2 mM Gln, rhGH decreased Gln net uptake 84% (1,096 +/- 215 to 155 +/- 150 nmol/4 h, P < 0.05), with basolateral uptake reversing to release; Glu- formation increased as ammonium production decreased. Monolayers grown on porous supports required rhGH to generate a transepithelial proton gradient within 4 h (0.01 +/- 0.05 control vs. 0.27 +/- 0.11 U for rhGH, P < 0.05); in addition, rhGH increased acid production (16.7 +/- 2.0 to 21.5 +/- 0.6 mumol x 4 h-1 x mg protein-1, P < 0.05). Adding insulin-like growth factor I (IGF-I) to the basolateral media decreased pH and enhanced Glu- and Gln uptake from that compartment.(ABSTRACT TRUNCATED AT 250 WORDS)
Lipoprotein lipase binding to adipocytes: evidence for the presence of a heparin-sensitive binding protein
Authors: Sasaki A. , Sivaram P. , Goldberg I. J.
Published: December 2017

Lipoprotein lipase (LPL) is synthesized by adipocytes, associated with the cell surface, and released from the cells when they are treated with heparin. Release of LPL from the adipocyte is required for LPL to migrate to its physiological site of action on the luminal surface of capillary endothelial cells. To better understand this process, we studied the interaction of LPL with adipocyte cell membrane proteins. With the use of a ligand blot method, LPL specifically bound to a heparin-releasable, 116-kDa protein on mouse-derived brown fat adipose cell (BFC-1 beta) and rat adipocyte membranes. A 116-kDa cell surface protein was metabolically labeled with [35S]methionine and bound to LPL-Sepharose. This suggested that the LPL-binding protein was synthesized by the cells. When BFC-1 beta were treated with heparin to eliminate heparin-sensitive cell surface binding sites, LPL binding to the cells decreased and release of newly synthesized LPL activity increased. 125I-labeled LPL binding to control cells was reduced (> 70%) by a 50-fold excess of unlabeled LPL. The residual LPL binding to heparin-treated cells was, however, not decreased by the addition of unlabeled LPL. These data imply that specific adipocyte surface LPL binding involves heparin-sensitive sites. We hypothesize that the heparin-releasable, 116-kDa LPL-binding protein mediates specific LPL binding to adipocytes and that LPL activity within adipose tissue is regulated, in part, by the interaction of LPL with this binding protein.
Linear V1-vascular vasopressin antagonists suitable for radioiodination, biotinylation, and fluorescent labeling
Authors: Thibonnier M. , Bayer A. L. , Madhun Z.
Published: December 2017

We modified several linear V1-vascular arginine vasopressin (AVP) antagonists to obtain compounds suitable for radioiodination, biotinylation, and fluorescent labeling. In binding competition experiments with human platelet V1-vascular AVP receptors, the linear V1 antagonist phenylacetyl-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2 (PhaaGln) displayed the greatest affinity [dissociation constant (Kd) = 0.05 +/- 0.01 nM]. The radioiodinated compound phenylacetyl-D-Tyr(Et)-Phe-Val-Asn-Lys-Pro-125I-labeled Tyr-NH2 (125I-labeled TyrPhaa) was characterized by a high affinity (Kd = 1.42 +/- 0.19 nM), a low nonspecific binding, and good stability. PhaaGln coupled to dodecabiotin retained a good affinity for V1-vascular AVP receptors (Kd = 1.41 +/- 0.20 nM). The complex PhaaGln-dodecabiotin-avidin is bifunctional, since an avidin-agarose column specifically bound V1-vascular AVP receptors labeled with 125I-TyrPhaa-dodecabiotin. In A7r5 vascular smooth muscle cells loaded with fura-2, PhaaGln and PhaaGln-dodecabiotin were pure antagonists as they blocked AVP-induced calcium mobilization but did not elicit a calcium signal by themselves. V1-vascular AVP receptors of A7r5 vascular smooth muscle cells were visualized by bound PhaaGln-dodecabiotin made fluorescent by labeling with fluorescein avidin. Thus linear V1-vascular AVP antagonists can be used as high affinity and specificity radioiodinated, biotinylated, and fluorescent probes to explore V1-vascular AVP receptors of human and animal origin.
Rat Prl and TSH secretion are regulated differently by K(+)-channel blockers
Authors: Wang X. , Inukai T. , Greer M. A. , Greer S. E.
Published: December 2017

All four different K(+)-channel blockers [tetraethylammonium (TEA), a nonselective K(+)-channel blocker; tolbutamide, an ATP-sensitive K(+)-channel blocker; quinine and 4-aminopyridine, both primarily voltage-dependent K(+)-channel blockers] stimulated prolactin (Prl) secretion by acutely dispersed anterior pituitary cells but had no effect on thyroid-stimulating hormone (TSH) secretion. TEA stimulated Prl secretion in a dose-dependent manner between 1 microM and 20 mM, but even as high as 20 mM, TEA did not induce TSH secretion. Valinomycin (2 microM), a K+ ionophore, inhibited both basal and TEA-induced Prl secretion. TEA-stimulated Prl secretion was abolished by using a Ca(2+)-depleted medium or adding 10 microM dopamine. TEA did not reverse the inhibitory effect of dopamine on thyrotropin-releasing hormone-induced Prl secretion. Our data indicate that K+ channels may play a role in the secretion of adenohypophysial hormones that is idiosyncratic for each hormone. Differences in the role of K+ channels may reflect differences between the various pituitary cell types in plasma membrane ion channel composition, membrane potential, or the mechanism of exocytosis.
Effects of selective disruption of cytoskeletal elements on steroid secretion by human adrenocortical slices
Authors: Feuilloley M. , Contesse V. , Lefebvre H. , Delarue C. , Vaudry H.
Published: December 2017

The role of the cytoskeleton in corticosteroid secretion in normal human adrenal gland was investigated in vitro, using the perifusion technique and confocal laser scanning microscopy. Vinblastine, which selectively disrupted microtubules in adrenocortical cells, did not modify the basal release of cortisol but induced a 58% inhibition of the response to adrenocorticotropic hormone (ACTH). In contrast, vinblastine did not alter dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-induced steroid secretion. Cytochalasin B treatment caused disappearance of microfilaments and blocked the stimulatory action of ACTH and DBcAMP on cortisol secretion. beta,beta'-Iminodipropionitrile disorganized the cytokeratin intermediate filaments but did not alter spontaneous and ACTH-evoked cortisol release. These results, which provide the first evidence for an action of cytoskeleton inhibitors on cortisol release from normal human adrenocortical cells, show that microtubules are involved in the mechanism of action of ACTH at a step preceding adenosine 3',5'-cyclic monophosphate formation, whereas microfilaments are involved in a late and common step of adrenal steroidogenesis.
Plasma vitamin C affects glucose homeostasis in healthy subjects and in non-insulin-dependent diabetics
Authors: Paolisso G. , D'Amore A. , Balbi V. , Volpe C. , Galzerano D. , Giugliano D. , Sgambato S. , Varricchio M. , D'Onofrio F.
Published: December 2017

In aged healthy (n = 10) and non-insulin-dependent (type II) diabetic (n = 10) subjects matched for age [67.3 +/- 0.5 vs. 68.0 +/- 0.4 yr, P = not significant (NS)], body mass index (25.7 +/- 0.7 vs. 26.0 +/- 0.2 kg/m2, P = NS), gender ratio [6 males (M)/4 females (F) vs. 5 M/5 F], and mean arterial blood pressure (104 +/- 6 vs. 105 +/- 9 mmHg, P = NS), we determined the changes in insulin secretion and action after vitamin C infusion and the relative increase in plasma vitamin C levels. At the highest vitamin C infusion rate (0.9 mmol/min) the increase in plasma vitamin C levels did not affect B cell response to glucose, but it improved Conard's K values and whole body glucose disposal in healthy subjects and in diabetic patients. In both groups of subjects vitamin C-mediated increase in insulin action was mainly due to an improvement in nonoxidative glucose metabolism. After fasting, plasma vitamin C levels correlated with basal whole body glucose disposal (r = -0.44, P < 0.05; n = 20). After vitamin C infusion, percent change in plasma vitamin C level correlated with the percent decline in membrane microviscosity (r = 0.53, P < 0.01; n = 20) and increase in whole body glucose disposal (r = 0.63, P < 0.003; n = 20). In conclusion, plasma vitamin C levels seem to play a role in the modulation of insulin action in aged healthy and diabetic subjects.
Lactate-pyruvate interconversion in blood: implications for in vivo tracer studies
Authors: Romijn J. A. , Chinkes D. L. , Schwarz J. M. , Wolfe R. R.
Published: December 2017

We have evaluated lactate and pyruvate kinetics in whole blood or plasma by the addition of [1-13C]lactate (n = 5) or [1-13C]pyruvate (n = 5) and application of compartmental modeling to the resulting data. Pyruvate and lactate concentrations and tracer-to-tracee ratios were measured at frequent intervals for 45 min. Pyruvate and lactate tracer-to-tracee ratios equilibrated almost completely within 3-4 min in whole blood, whereas there was no isotopic exchange in plasma. The average rate of interconversion between unlabeled lactate and pyruvate was four to five times (pyruvate to lactate) and three to four times (lactate to pyruvate) the net production rate of lactate. We conclude that there is a very rapid interconversion between lactate and pyruvate in blood that has to be considered in the interpretation of in vivo tracer studies.
Type II glucocorticoid receptors in the CNS regulate metabolism in ob/ob mice independent of protein synthesis
Authors: Chen H. L. , Romsos D. R.
Published: December 2017

A single intracerebroventricular injection of dexamethasone rapidly (within 30 min) decreases brown adipose tissue thermogenesis by 25% as assessed by GDP binding and increases plasma insulin twofold in adrenalectomized ob/ob mice. The present study investigated the type of corticoid receptor(s) that mediate these effects and determined whether protein synthesis was necessary for expression of these glucocorticoid actions in ob/ob mice. Intracerebroventricular injection of aldosterone (a type I-corticoid receptor agonist) was ineffective in altering peripheral metabolism in adrenalectomized ob/ob mice, whereas RU-486 (a type II-corticoid receptor antagonist) abolished the effects of dexamethasone. Thus type II-like corticoid receptors, not type I receptors, mediated the rapid effects of dexamethasone in adrenalectomized ob/ob mice. Anisomycin (0.5 mg) administered subcutaneously almost completely suppressed (-92%) cerebral protein synthesis, but anisomycin did not abolish the rapid effects of dexamethasone in adrenalectomized ob/ob mice. Thus protein synthesis is not a prerequisite for rapid effects of dexamethasone in adrenalectomized ob/ob mice.
Trypsin-Mn(2+)-resistant form of type 1 protein phosphatase in human muscle
Authors: Mori H. , Stone K. , Mott D. M.
Published: December 2017

Reduced type 1 protein phosphatase (PP-1) activity in human muscle extracts may contribute to the reduced insulin-stimulated glycogen synthase activity associated with insulin resistance for glucose disposal in humans. Because inactive forms of PP-1 can be activated with trypsin plus Mn2+, these reagents were used to compare the PP-1 activities in skeletal muscle extracts before and after separation into cytosolic and glycogen microsomal (GM) fractions. PP-1 activities were reduced in the GM fraction from insulin-resistant subjects (54 +/- 2 vs. 61 +/- 1, P < 0.01) but, in contrast to our previously published results, were elevated in the extract (33 +/- 6 vs. 18 +/- 3, P < 0.05). Recombination of the cytosol and GM fractions (reconstituted extract) demonstrated that the low extract PP-1 activities could only be regenerated when the GM fraction from insulin-sensitive subjects was recombined with cytosol from either group. The results indicate that the elevated PP-1 activity observed in extracts of insulin-resistant compared with insulin-sensitive subjects is caused by an inhibitor of extract PP-1 activity that sediments with the GM pellet and is more active in the insulin-sensitive subjects.
Disposition of a mixed meal by the conscious dog
Authors: Moore M. C. , Pagliassotti M. J. , Swift L. L. , Asher J. , Murrell J. , Neal D. , Cherrington A. D.
Published: December 2017

The disposition of a mixed meal administered intragastrically was examined in 13 24-h-fasted conscious dogs, using the arteriovenous (AV) difference technique (and isotopic methods in 6 dogs). Postprandial net gut output totaled (in g of glucose equivalents) 42 +/- 6 glucose, 3 +/- 0.3 lactate, 2 +/- 0.2 alanine, and 0.2 +/- 0.0 glycerol. The gut oxidized 2 +/- 1 g of glucose, and 0.2 +/- 0.1 g remained within the intestinal lumen. Of the administered glucose 68 +/- 6% were accounted for, and volatile fatty acid production by the gut (n = 1) accounted for at least an additional 4%. Of the labeled glucose in the meal 82 +/- 5% appeared in the systemic circulation, an apparent overestimate of absorption of glucose from the meal. Cumulative net hepatic uptakes (in g of glucose equivalents) were 4.1 +/- 3.1 glucose, 12.1 +/- 2.1 gluconeogenic amino acids, and 1.5 +/- 0.2 glycerol. Net hepatic glycogen synthesis and lactate and CO2 production accounted for 6.2 +/- 4.1, 9.3 +/- 2.8, and 1.6 +/- 0.8 g of glucose equivalents, respectively. In summary, the AV difference method could account for the gut disposition of about two-thirds of the meal glucose. Nonsplanchnic tissues disposed of threefold more glucose than the liver. Net hepatic uptake of glucose equivalents as gluconeogenic amino acids was threefold > glucose uptake, and net hepatic uptake of gluconeogenic amino acids was > net gut release of gluconeogenic amino acids. In conclusion, the net hepatic uptake of glucose and gluconeogenic substrates provided adequate carbon for net hepatic synthesis of glycogen and production of lactate and CO2. In a net sense, peripheral tissues must have been the source of some of the gluconeogenic carbon taken up by the liver after the meal.
Hypoxic effect of exogenous insulin on normal and diabetic peripheral nerve
Authors: Kihara M. , Zollman P. J. , Smithson I. L. , Lagerlund T. D. , Low P. A.
Published: December 2017

Insulin administration can cause or worsen experimental and human diabetic neuropathy ("insulin neuritis"). In this study, we tested the hypothesis that insulin administration impairs tissue oxygenation. We infused insulin under nonhypoglycemic conditions and evaluated its effect on endoneurial oxygen tension, nerve blood flow, and the oxyhemoglobin dissociation curve of peripheral nerve in normal and diabetic rats. Intravenous insulin infusion resulted in a dose-dependent reduction in endoneurial oxygen tension in normal nerves (from 26% at 0.04 U/kg insulin to 55% at 32 U/kg). The nerves of rats with streptozotocin-induced diabetes were resistant, but with control of hyperglycemia this susceptibility to the endoneurial hypoxic effect of insulin returned. The reduction in endoneurial oxygen tension regressed with glycosylated hemoglobin (Y = 53.8-2.7X, where Y = %reduction in endoneurial oxygen tension and X = HbA1; r = 0.87; P = < 0.001). Diabetes or insulin administration resulted in only minimal and physiologically insignificant alterations in the oxygen dissociation curve and 2,3-diphosphoglycerate of sciatic nerve. Instead, insulin administration resulted in a reduction in nerve nutritive blood flow and an increase in arteriovenous shunt flow. When the latter was eliminated by the closure of arteriovenous shunts (infusion of 5-hydroxytryptamine), endoneurial oxygen reverted to normal. These findings indicate a deleterious vasoactive effect of insulin and may explain the development of insulin neuritis.
Response of protein synthesis in human skeletal muscle to insulin: an investigation with L-[2H5]phenylalanine
Authors: McNurlan M. A. , Essen P. , Thorell A. , Calder A. G. , Anderson S. E. , Ljungqvist O. , Sandgren A. , Grant I. , Tjader I. , Ballmer P. E. , et al.
Published: December 2017

The role of insulin in the regulation of muscle protein synthesis in adult humans has been investigated with intravenous infusion of insulin at levels comparable with those observed after normal feeding. Glucose was also infused to maintain euglycemia. Muscle protein synthesis was measured in six healthy subjects before and during insulin and glucose infusion from the incorporation of L-[2H5]phenylalanine into the protein of vastus lateralis sampled by percutaneous biopsy. L-[2H5]phenylalanine was given as a single injection of a flooding amount (45 mg/kg). The relatively low levels of enrichment of phenylalanine in protein (0.005 atom%) were measured by modified gas chromatography-mass spectrometry and verified by comparison with incorporation of L-[2,6-3H]phenylalanine. Similarity of enrichment in tissue-free and plasma pools (flooding) and linear incorporation over the period of measurement were also verified. The fractional rate of muscle protein synthesis in the group of postabsorptive subjects was 1.65 +/- 0.11% (SE)/day. The rate was unaltered by insulin and glucose infusion, 1.66 +/- 0.16%/day.