bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermal instability of rat muscle sarcoplasmic reticulum Ca2+-ATPase function
Authors: Schertzer J. D. , Green H. J. , Tupling A. R.
Published: February 2015

To examine the thermal instability and the role of sulfhydryl (SH) oxidation on sarcoplasmic reticulum (SR) Ca2+-ATPase function, crude homogenates were prepared from the white portion of the gastrocnemius (WG) adult rat muscles ( n = 9) and incubated in vitro for ≤60 min either at a normal resting body temperature (37°C) or at a temperature indicative of exercise-induced hyperthermia (41°C) with DTT and without DTT (CON). In general, treatment with DTT resulted in higher Ca2+-ATPase and Ca2+uptake values (nmol · mg protein−1· min−1, P < 0.05), an effect that was not specific to time of incubation. Incubations at 41°C resulted in lower ( P< 0.05) Ca2+uptake rates (156 ± 18 and 35.9 ± 3.3) compared with 37°C (570 ± 54 and 364 ± 26) at 30 and 60 min, respectively. At 37°C, ryanodine (300 μM), which was used to block Ca2+release from the calcium release channel, prevented the time-dependent decrease in Ca2+uptake. A general inactivation ( P < 0.05) of maximal Ca2+-ATPase activity ( Vmax) in CON was observed with incubation time (0 > 30 > 60 min), with the effect being more pronounced ( P < 0.05) at 41°C compared with 37°C. The Hill slope, a measure of co-operativity, and the pCa50, the cytosolic Ca2+concentration required for half-maximal activation of Ca2+-ATPase activity, decreased ( P < 0.05) at 41°C only. Treatment with DTT attenuated the alterations in enzyme kinetics. The increase in Vmaxwith the Ca2+ionophore A-23187 was less pronounced at 41°C compared with 37°C. It is concluded that exposure of homogenates to a temperature typically experienced in exercise results in a reduction in the coupling ratio, which is mediated primarily by lower Ca2+uptake and occurs as a result of increases in membrane permeability to Ca2+. Moreover, the decreases in Ca2+-ATPase kinetics in WG with sustained heat stress result from SH oxidation.
Aminoacyl-tRNA enrichment after a flood of labeled phenylalanine: insulin effect on muscle protein synthesis
Authors: Caso Giuseppe , Ford G. Charles , Nair K. Sreekumaran , Garlick Peter J. , McNurlan Margaret A.
Published: February 2015

Muscle protein synthesis in dogs measured by flooding withl-[2H5]phenylalanine (70 mg/kg) was significantly stimulated by infusion of insulin with amino acids. The stimulation of muscle protein synthesis was similar when calculated from the enrichment of phenylalanyl-tRNA (61 ± 10%, P< 0.001), plasma phenylalanine (61 ± 10%, P < 0.001), or tissue fluid phenylalanine (54 ± 10%, P < 0.001). The time course for changes in enrichment ofl-[2H5]phenylalanine throughout the flooding period was determined for plasma, tissue fluid, and phenylalanyl-tRNA in the basal state and during the infusion of insulin with amino acids. Enrichments of plasma free phenylalanine and phenylalanyl-tRNA were equalized between 20 and 45 min, although the enrichment of phenylalanyl-tRNA was lower at early time points. Rates of muscle protein synthesis obtained with the flooding method and calculated from plasma phenylalanine enrichment were comparable to those calculated from phenylalanyl-tRNA and also to those obtained previously with a continuous infusion of phenylalanine with phenylalanyl-tRNA as precursor. This study confirms that, with a bolus injection of labeled phenylalanine, the enrichment of aminoacyl-tRNA, the true precursor pool for protein synthesis, can be assessed from more readily sampled plasma phenylalanine.
ACTH-induced Cl−current in bovine adrenocortical cells: correlation with cortisol secretion
Authors: Dupré-Aucouturier Sylvie , Penhoat Armelle , Rougier Oger , Bilbaut André
Published: February 2015

ACTH has been shown to depolarize bovine adrenal zona fasciculata cells by inhibiting a K+current. The effects of this hormone on such cells have been reexamined using perforated and standard patch recording methods. In current clamp experiments, ACTH (10 nM) induced a membrane depolarization to −36 ± 1 mV ( n = 56), which was mimicked by forskolin (10 μM) or by 8-(4-chlorophenylthio)-cAMP (8 mM). ACTH-induced membrane depolarizations were associated in the majority of cells with an increase in membrane conductance. In the other cells, these membrane responses could occur without change or could be correlated with a transient or with a continuous Cs+-sensitive decrease in membrane conductance. The depolarizations associated with an increase in membrane conductance were depressed by Cl−current inhibitors diphenylamine-2-carboxylic acid (DPC; 1 mM), anthracene-9-carboxylic acid (9-AC; 1 mM), DIDS (400 μM), verapamil (100 μM), and glibenclamide (20 μM). In voltage-clamped Cs+-loaded cells, ACTH activated a time-independent current that displayed an outward rectification and reversed at −21.5 mV ± 2 ( n = 6). This current, observed in the presence of internal EGTA (5 mM), was depressed in low Cl−external solution and was inhibited by DPC, 9-AC, DIDS, 5-nitro-2-(3-phenylpropylamino)benzoic acid, verapamil, and glibenclamide. ACTH-stimulated cortisol secretion was blocked by Cl−channel inhibitors DIDS (400 μM) and DPC (1 mM). The present results reveal that, in addition to inhibiting a K+current, ACTH activates in bovine zona fasciculata cells a Ca2+-insensitive, cAMP-dependent Cl−current. This Cl−current is involved in the ACTH-induced membrane depolarization, which seems to be a crucial step in stimulating steroidogenesis.
Impact of chronic fructose infusion on hepatic metabolism during TPN administration
Authors: Donmoyer Christine M. , Lacy D. Brooks , Zhang Yiqun , Chen Sheng-Song , McGuinness Owen P.
Published: February 2015

During chronic total parenteral nutrition (TPN), net hepatic glucose uptake (NHGU) is markedly elevated. However, NHGU is reduced by the presence of an infection. We recently demonstrated that a small, acute (3-h) intraportal fructose infusion can correct the infection-induced impairment in NHGU. The aim of this study was to determine whether the addition of fructose to the TPN persistently enhances NHGU in the presence of an infection. TPN was infused continuously into the inferior vena cava of chronically catheterized dogs for 5 days. On day 3, a bacterial clot was implanted in the peritoneal cavity, and either saline (CON, n = 5) or fructose (+FRUC, 1.0 mg · kg−1· min−1, n = 6) infusion was included with the TPN. Forty-two hours after the infection was induced, hepatic glucose metabolism was assessed in conscious dogs with arteriovenous and tracer methods. Arterial plasma glucose concentration was lower with chronic fructose infusion (120 ± 4 vs. 131 ± 3 mg/dl, +FRUC vs. CON, P < 0.05); however, NHGU was not enhanced (2.2 ± 0.5 vs. 2.8 ± 0.4 mg · kg−1· min−1). Acute removal of the fructose infusion dramatically decreased NHGU (2.2 ± 0.5 to −0.2 ± 0.5 mg · kg−1· min−1), and net hepatic lactate release also fell (1.6 ± 0.3 to 0.5 ± 0.3 mg · kg−1· min−1). This led to an increase in the arterial plasma glucose (Δ13 ± 3 mg/dl, P < 0.05) and insulin (Δ5 ± 2 μU/ml) concentrations and to a decrease in glucagon (Δ−11 ± 3 pg/ml) concentration. In conclusion, the addition of chronic fructose infusion to TPN during infection does not lead to a persistent augmentation of NHGU.
Evidence of a malonyl-CoA-insensitive carnitine palmitoyltransferase I activity in red skeletal muscle
Authors: Kim Jong-Yeon , Koves Timothy R. , Yu Geng-Sheng , Gulick Tod , Cortright Ronald N. , Dohm G. Lynis , Muoio Deborah M.
Published: February 2015

Carnitine palmitoyltransferase I (CPT I), which is expressed as two distinct isoforms in liver (α) and muscle (β), catalyzes the rate-limiting step in the transport of fatty acid into the mitochondria. Malonyl-CoA, a potent inhibitor of CPT I, is considered a key regulator of fatty acid oxidation in both tissues. Still unanswered is how muscle β-oxidation proceeds despite malonyl-CoA concentrations that exceed the IC50for CPT Iβ. We evaluated malonyl-CoA-suppressible [14C]palmitate oxidation and CPT I activity in homogenates of red (RG) and white (WG) gastrocnemius, soleus (SOL), and extensor digitorum longus (EDL) muscles. Adding 10 μM malonyl-CoA inhibited palmitate oxidation by 29, 39, 60, and 89% in RG, SOL, EDL, and WG, respectively. Thus malonyl-CoA resistance, which correlated strongly (0.678) with absolute oxidation rates (RG > SOL > EDL > WG), was greater in red than in white muscles. Similarly, malonyl-CoA-resistant palmitate oxidation and CPT I activity were greater in mitochondria from RG compared with WG. Ribonuclease protection assays were performed to evaluate whether our data might be explained by differential expression of CPT I splice variants. We detected the presence of two CPT Iβ splice variants that were more abundant in red compared with white muscle, but the relative expression of the two mRNA species was unrelated to malonyl-CoA resistance. These results provide evidence of a malonyl-CoA-insensitive CPT I activity in red muscle, suggesting fiber type-specific expression of distinct CPT I isoforms and/or posttranslational modulations that have yet to be elucidated.
Reactive oxygen species mediate leukocyte-endothelium interactions in prostaglandin F2α-induced luteolysis in rats
Authors: Minegishi Kazuhiro , Tanaka Mamoru , Nishimura Osamu , Tanigaki Shinji , Miyakoshi Kei , Ishimoto Hitoshi , Yoshimura Yasunori
Published: February 2015

We investigated the contribution of neutrophils to prostaglandin (PG)F2α-induced luteolysis and the role of reactive oxygen species (ROS) as potential mediators of neutrophil accumulation in regressing corpora lutea of superovulated rats. On day 8of pseudopregnancy, subcutaneous injection of PGF2α(500 μg) significantly increased rhodamine 6G-labeled leukocyte adhesion in luteal venules, as observed by intravital microscopy. Neutrophil accumulation was confirmed by significantly increased myeloperoxidase (MPO) activity. Pretreatment with anti-leukocyte antibody (CD18-directed monoclonal antibody, WT-3) significantly inhibited the PGF2α-induced increases in adherent leukocytes and MPO activity. Anti-leukocyte antibody also maintained serum progesterone concentrations. Pretreatment with oxygen free radical scavengers, superoxide dismutase (50,000 U/kg) and catalase (90,000 U/kg), also attenuated these PGF2α-induced alterations. Corpora lutea preloaded with dichlorodihydrofluorescein diacetate succinimidyl ester, a fluorescent indicator for determining intracellular ROS generation, exhibited an increase in fluorescence after PGF2αtreatment. These findings suggest that leukocyte-endothelium interactions mediated by ROS generation are important in PGF2α-induced luteolysis in rats.
1α,25(OH)2-vitamin D3inhibits HGF synthesis and secretion from MG-63 human osteosarcoma cells
Authors: Chattopadhyay Naibedya , MacLeod R. J. , Tfelt-Hansen Jacob , Brown Edward M.
Published: February 2015

Several mesenchymally derived cells, including osteoblasts, secrete hepatocyte growth factor (HGF). 1α,25(OH)2-vitamin D3[1,25(OH)2D3] inhibits proliferation and induces differentiation of MG-63 osteoblastic cells. Here we show that MG-63 cells secrete copious amounts of HGF and that 1,25(OH)2D3inhibits HGF production. MG-63 cells also express HGF receptor (c-Met) mRNA, suggesting an autocrine action of HGF. Indeed, although exogenous HGF failed to stimulate cellular proliferation, neutralizing endogenous HGF with a neutralizing antibody inhibited MG-63 cell proliferation; moreover, inhibiting HGF synthesis with 1,25(OH)2D3followed by addition of HGF rescued hormone-induced inhibition of proliferation. Nonneutralized cells displayed constitutive phosphorylation of c-Met and the mitogen-activated protein kinases mitogen/extracellular signal-regulated kinase (MEK) 1 and extracellular signal-regulated kinase (Erk) 1/2, which were inhibited by anti-HGF antibody. Constitutive phosphorylation of Erk1/2 was also abolished by 1,25(OH)2D3. Addition of HGF to MG-63 cells treated with neutralizing HGF antibody induced rapid phosphorylation of c-Met, MEK1, and Erk1/2. Thus endogenous HGF induces a constitutively active, autocrine mitogenic loop in MG-63 cells. The known antiproliferative effect of 1,25(OH)2D3on MG-63 cells can be accounted for by the concomitant 1,25(OH)2D3-induced inhibition of HGF production.
Fetal hepatic and umbilical uptakes of glucogenic substrates during a glucagon-somatostatin infusion
Authors: Teng Cecilia , Battaglia Frederick C. , Meschia Giacomo , Narkewicz Michael R. , Wilkening Randall B.
Published: February 2015

To test the hypothesis that fetal hepatic glutamate output diverts the products of hepatic amino acid metabolism from hepatic gluconeogenesis, ovine fetal hepatic and umbilical uptakes of glucose and glucogenic substrates were measured before and during fetal glucagon-somatostatin (GS) infusion and during the combined infusion of GS, alanine, glutamine, and arginine. Before the infusions, hepatic uptake of lactate, alanine, glutamine, arginine, and other substrates was accompanied by hepatic output of pyruvate, aspartate, serine, glutamate, and ornithine. The GS infusion induced hepatic output of 1.00 ± 0.07 mol glucose carbon/mol O2uptake, an equivalent reduction in hepatic output of pyruvate and glutamate carbon, a decrease in umbilical glucose uptake and placental uptake of fetal glutamate, an increase in hepatic alanine and arginine clearances, and a decrease in umbilical alanine, glutamine, and arginine uptakes. The latter result suggests that glucagon inhibits umbilical amino acid uptake. We conclude that fetal hepatic pyruvate and glutamate output is part of an adaptation to placental function that requires the fetal liver to maintain both a high rate of catabolism of glucogenic substrates and a low rate of gluconeogenesis.
Parathyroid gland volume increases with postmaturational aging in the rat
Authors: Halloran Bernard , Udén Per , Duh Quan-Yang , Kikuchi Shoichi , Wieder Tracy , Cao Jay , Clark Orlo
Published: February 2015

To examine the pathophysiology of the age-related rise in the plasma concentration of parathyroid hormone (PTH), we studied the relationships among plasma immunoreactive PTH (iPTH), parathyroid gland volume, parathyroid cell proliferation rate, renal function, and blood Ca2+ in male Fischer 344 rats aged 6–28 mo. Plasma iPTH increased 2.5-fold between 6 and 28 mo and correlated with parathyroid gland volume ( r = 0.87). Gland volume began to increase as early as 6–12 mo of age and by 28 mo was threefold greater than at 6 mo. Gland expansion was a consequence of hyperplasia stimulated in part by an increase in cell proliferative activity late in life. Blood Ca2+ and plasma inorganic phosphorus did not change significantly with age. Glomerular filtration rate decreased with age but only after the age of 24 mo. Unlike what has been observed in the human, these data suggest that the age-related increase in plasma iPTH in the rat is linked to parathyroid gland hyperplasia and that early gland growth does not appear to be associated with hypocalcemia or renal insufficiency, but rather to developmentally related metabolic changes. Later in life (>24 mo), the increase in parathyroid cell proliferation rate, further hyperplastic expansion of the gland, and increase in iPTH secretion appear to be associated with renal insufficiency.
Pattern and cost of weight gain in previously obese women
Authors: Votruba S. B. , Blanc S. , Schoeller D. A.
Published: February 2015

Weight gain is common among postobese individuals, providing an opportunity to address the cost of weight regain on energy expenditure. We investigated the energy cost of weight regain over 1 yr in 28 women [age 39.5 ± 1.3 (SE) yr; body mass index 24.2 ± 0.5 kg/m2] with recent weight loss (>12 kg). Body composition, total energy expenditure (TEE) using doubly labeled water, resting metabolic rate (RMR), and thermic effect of a meal (TEM) were assessed at 0 and 12 mo. Metabolizable energy intake (MEI) was calculated from TEE and change in body composition. Fourteen women had a weight gain of 13.2 ± 2.1 kg. Twelve-month cumulative excess MEI, calculated as the intake in excess of TEE at month 0, was 749 ± 149 MJ. Of this, 462 ± 83 MJ (62%) were stored as accrued tissue, and 287 ± 72 MJ (38%) was increased TEE. Expressed per kilogram of body weight gain, the energy cost of weight gain was calculated to be 54.8 ± 4.6 MJ/kg. Interestingly, weight regain time courses fell into three distinct patterns, possibly requiring varying countermeasures.
Developmental decline in components of signal transduction pathways regulating protein synthesis in pig muscle
Authors: Kimball Scot R. , Farrell Peter A. , Nguyen Hahn V. , Jefferson Leonard S. , Davis Teresa A.
Published: February 2015

Our previous studies showed that the feeding-induced stimulation of protein synthesis in skeletal muscle of neonatal pigs is accompanied by enhanced phosphorylation of the eukaryotic initiation factor (eIF)4E-binding protein (4E-BP1) and the ribosomal protein S6 kinase (S6K1). These effects of feeding are substantially reduced with development. The goal of the present investigation was to delineate the basis for the reduced responsiveness to feeding observed in the older animals. In these studies, the content and activity of protein kinases located upstream of S6K1 and 4E-BP1 in signal transduction pathways activated by amino acids, insulin, and insulin-like growth factor I were examined in 7- and 26-day-old pigs that were either fasted overnight or fed porcine milk after an overnight fast. Feeding stimulated phosphatidylinositol (PI) 3-kinase activity to the same extent in muscle of 7- and 26-day-old pigs, suggesting that PI 3-kinase is not limiting in muscle of older animals. In contrast, protein kinase B (PKB) activity was significantly less in muscle from 26- vs. 7-day-old pigs, regardless of nutritional status, suggesting that its activity is regulated by mechanisms distinct from PI 3-kinase. In part, the reduced PKB responsiveness can be attributed to a developmental decline in PKB content. Likewise, muscle content of the protein kinase termed mammalian target of rapamycin (mTOR) in 26-day-old pigs was <25% of that in 7-day-old animals. Finally, in agreement with our earlier work showing that S6K1 phosphorylation is reduced in older animals, S6K1 activity was stimulated to a lesser extent in 26- compared with 7-day-old pigs. Overall, the results suggest that the blunted protein synthetic response observed in 26- vs. 7-day-old neonatal pigs is due in part to decreased content and/or activity of signaling components downstream of PI 3-kinase, e.g., PKB, mTOR, and S6K1.
β-Oxidation of free fatty acids is required to maintain translational control of protein synthesis in heart
Authors: Crozier Stephen J. , Bolster Douglas R. , Reiter Ali K. , Kimball Scot R. , Jefferson Leonard S.
Published: February 2015

The study described herein investigated the role of free fatty acids (FFAs) in the maintenance of protein synthesis in vivo in rat cardiac and skeletal muscle. Suppression of FFA β-oxidation by methyl palmoxirate caused a marked reduction in protein synthesis in the heart. The effect on protein synthesis was mediated in part by changes in the function of eukaryotic initiation factors (eIFs) involved in the initiation of mRNA translation. The guanine nucleotide exchange activity of eIF2B was repressed, phosphorylation of the α-subunit of eIF2 was enhanced, and phosphorylation of eIF4E-binding protein-1 and ribosomal protein S6 kinase was reduced. Similar changes in protein synthesis and translation initiation were not observed in the gastrocnemius following treatment with methyl palmoxirate. In heart, repressed β-oxidation of FFA correlated, as demarcated by changes in the ATP/AMP ratio and phosphorylation of AMP-activated kinase, with alterations in the energy status of the tissue. Therefore, the activation state of signal transduction pathways that are responsive to cellular energy stress represents one mechanism whereby translation initiation may be regulated in cardiac muscle.
Retinoic acid is detected at relatively high levels in the CNS of adult rats
Authors: Werner Elizabeth A. , Deluca Hector F.
Published: February 2015

Retinoic acid (RA) is essential for cellular growth and differentiation in developing and adult animals. The central nervous system (CNS) suffers developmental defects if embryonic levels of RA are too high or too low. The production and function of RA in adult brain are unclear. We report that RA is present throughout the brain and spinal cord of adult, vitamin A-deficient (VAD) rats treated with a physiological amount of all- trans-retinol. The hippocampus/cortex contained the highest proportion of RA in the brain (27.2 ± 2.9% of the organic phase radioactivity, and 23.5 ± 0.8% of the organic phase radioactivity extracted from spinal cord was RA). RA comprises a higher proportion of the retinoid pool in the CNS compared with amounts reported in other target tissues (E Werner and HF DeLuca. Arch Biochem Biophys 393: 262–270, 2001). However, RA is not preferentially transported from the blood to the brain. There were 2.90 ± 0.20 fmol RA/g tissue transported to the brain of VAD rats treated with 2.00 nmol [20-3H]all- trans-retinoic acid, but higher amounts of RA were delivered to the liver, testis, and spleen. Because RA is not transported preferentially to brain, this tissue likely synthesizes RA more efficiently than other target tissues.
Fatty acid oxidation and triacylglycerol hydrolysis are enhanced after chronic leptin treatment in rats
Authors: Steinberg Gregory R. , Bonen Arend , Dyck David J.
Published: February 2015

Leptin acutely increases fatty acid (FA) oxidation and triacylglycerol (TG) hydrolysis and decreases TG esterification in oxidative rodent muscle. However, the effects of chronic leptin administration on FA metabolism in skeletal muscle have not been examined. We hypothesized that chronic leptin treatment would enhance TG hydrolysis as well as the capacity to oxidize FA in soleus (SOL) muscle. Female Sprague-Dawley rats were infused for 2 wk with leptin (LEPT; 0.5 mg · kg−1· day−1) by use of subcutaneously implanted miniosmotic pumps. Control (AD-S) and pair-fed (PF-S) animals received saline-filled implants. Subsequently, FA metabolism was monitored for 45 min in isolated, resting, and contracting (20 tetani/min) SOL muscles by means of pulse-chase procedures. Food intake (−33 ± 2%, P < 0.01) and body mass (−12.5 ± 4%, P = 0.01) were reduced in both LEPT and PF-S animals. Leptin levels were elevated (+418 ± 7%, P < 0.001) in treated animals but reduced in PF-S animals (−73 ± 8%, P< 0.05) relative to controls. At rest, TG hydrolysis was increased in leptin-treated rats (1.8 ± 2.2, AD-S vs. 23.5 ± 8.1 nmol/g wet wt, LEPT; P < 0.001). In contracting SOL muscles, TG hydrolysis (1.5 ± 0.6, AD-S vs. 3.6 ± 1.0 μmol/g wet wt, LEPT; P = 0.02) and palmitate oxidation (18.3 ± 6.7, AD-S vs. 45.7 ± 9.9 nmol/g wet wt, LEPT; P < 0.05) were both significantly increased by leptin treatment. Chronic leptin treatment had no effect on TG esterification either at rest or during contraction. Markers of overall (citrate synthase) and FA (hydroxyacyl-CoA dehydrogenase) oxidative capacity were unchanged with leptin treatment. Protein expression of hormone-sensitive lipase (HSL) was also unaltered following leptin treatment. Thus leptin-induced increases in lipolysis are likely due to HSL activation (i.e., phosphorylation). Increased FA oxidation secondary to chronic leptin treatment is not due to an enhanced oxidative capacity and may be a result of enhanced flux into the mitochondrion (i.e., carnitine palmitoyltransferase I regulation) or electron transport uncoupling (i.e., uncoupling protein-3 expression).
Partitioning glucose distribution/transport, disposal, and endogenous production during IVGTT
Authors: Hovorka Roman , Shojaee-Moradie Fariba , Carroll Paul V. , Chassin Ludovic J. , Gowrie Ian J. , Jackson Nicola C. , Tudor Romulus S. , Umpleby A. Margot , Jones Richard H.
Published: February 2015

We have separated the effect of insulin on glucose distribution/transport, glucose disposal, and endogenous production (EGP) during an intravenous glucose tolerance test (IVGTT) by use of a dual-tracer dilution methodology. Six healthy lean male subjects (age 33 ± 3 yr, body mass index 22.7 ± 0.6 kg/m2) underwent a 4-h IVGTT (0.3 g/kg glucose enriched with 3–6% d-[U-13C]glucose and 5–10% 3- O-methyl-d-glucose) preceded by a 2-h investigation under basal conditions (5 mg/kg ofd-[U-13C]glucose and 8 mg/kg of 3- O-methyl-d-glucose). A new model described the kinetics of the two glucose tracers and native glucose with the use of a two-compartment structure for glucose and a one-compartment structure for insulin effects. Insulin sensitivities of distribution/transport, disposal, and EGP were similar (11.5 ± 3.8 vs. 10.4 ± 3.9 vs. 11.1 ± 2.7 × 10−2ml · kg−1· min−1per mU/l; P = nonsignificant, ANOVA). When expressed in terms of ability to lower glucose concentration, stimulation of disposal and stimulation of distribution/transport accounted each independently for 25 and 30%, respectively, of the overall effect. Suppression of EGP was more effective ( P < 0.01, ANOVA) and accounted for 50% of the overall effect. EGP was suppressed by 70% (52–82%) (95% confidence interval relative to basal) within 60 min of the IVGTT; glucose distribution/transport was least responsive to insulin and was maximally activated by 62% (34–96%) above basal at 80 min compared with maximum 279% (116–565%) activation of glucose disposal at 20 min. The deactivation of glucose distribution/transport was slower than that of glucose disposal and EGP ( P < 0.02) with half-times of 207 (84–510), 12 (7–22), and 29 (16–54) min, respectively. The minimal-model insulin sensitivity was tightly correlated with and linearly related to sensitivity of EGP ( r = 0.96, P < 0.005) and correlated positively but nonsignificantly with distribution/transport sensitivity ( r = 0.73, P = 0.10) and disposal sensitivity ( r = 0.55, P = 0.26). We conclude that, in healthy subjects during an IVGTT, the two peripheral insulin effects account jointly for approximately one-half of the overall insulin-stimulated glucose lowering, each effect contributing equally. Suppression of EGP matches the effect in the periphery.
Salmon cardiac natriuretic peptide is a volume-regulating hormone
Authors: Tervonen Virpi , Ruskoaho Heikki , Lecklin Tiina , Ilves Mika , Vuolteenaho Olli
Published: February 2015

The present study tested the hypothesis that salmon cardiac peptide (sCP), a new member of the family of natriuretic peptides, has an important role in the regulation of fluid balance and cardiovascular function. Intra-arterial administration of sCP increased urine output in salmon. It had a diuretic effect in rat as well, but the potency was lower. sCP increased the sodium excretion in proportion to the increased urine flow. Blood pressure was not affected by sCP in either species. Acute volume expansion elevated the plasma level of sCP in salmon, and an acute transfer of salmon from fresh to sea water decreased the circulating sCP level. Cardiac immunoreactive sCP or sCP mRNA levels were not affected by transfer to sea water. These results indicate that sCP has an important physiological role in defending salmon against volume overload but that it does not appear to contribute to the short-term regulation of blood pressure. sCP provides an excellent model of the general mechanisms of regulation of the A-type (atrial) natriuretic peptide system.
Free fatty acid-induced peripheral insulin resistance augments splanchnic glucose uptake in healthy humans
Authors: Bajaj Mandeep , Berria Rachele , Pratipanawatr Thongchai , Kashyap Sangeeta , Pratipanawatr Wilailak , Belfort Renata , Cusi Kenneth , Mandarino Lawrence , DeFronzo Ralph A.
Published: February 2015

To investigate the effect of elevated plasma free fatty acid (FFA) concentrations on splanchnic glucose uptake (SGU), we measured SGU in nine healthy subjects (age, 44 ± 4 yr; body mass index, 27.4 ± 1.2 kg/m2; fasting plasma glucose, 5.2 ± 0.1 mmol/l) during an Intralipid-heparin (LIP) infusion and during a saline (Sal) infusion. SGU was estimated by the oral glucose load (OGL)-insulin clamp method: subjects received a 7-h euglycemic insulin (100 mU · m−2 · min−1) clamp, and a 75-g OGL was ingested 3 h after the insulin clamp was started. After glucose ingestion, the steady-state glucose infusion rate (GIR) during the insulin clamp was decreased to maintain euglycemia. SGU was calculated by subtracting the integrated decrease in GIR during the period after glucose ingestion from the ingested glucose load. [3-3H]glucose was infused during the initial 3 h of the insulin clamp to determine rates of endogenous glucose production (EGP) and glucose disappearance (Rd). During the 3-h euglycemic insulin clamp before glucose ingestion, Rd was decreased (8.8 ± 0.5 vs. 7.6 ± 0.5 mg · kg−1 · min−1, P < 0.01), and suppression of EGP was impaired (0.2 ± 0.04 vs. 0.07 ± 0.03 mg · kg−1 · min−1, P < 0.01). During the 4-h period after glucose ingestion, SGU was significantly increased during the LIP vs. Sal infusion study (30 ± 2 vs. 20 ± 2%, P < 0.005). In conclusion, an elevation in plasma FFA concentration impairs whole body glucose Rd and insulin-mediated suppression of EGP in healthy subjects but augments SGU.
Influence of oral contraceptive use on endothelial t-PA release in healthy premenopausal women
Authors: Hoetzer Greta L. , Stauffer Brian L. , Greiner Jared J. , Casas Yoli , Smith Derek T. , DeSouza Christopher A.
Published: February 2015

We determined the influence of oral contraceptives (OC) on the capacity of the endothelium to release tissue-type plasminogen activator (t-PA). Twenty-three healthy premenopausal women were studied: 12 nonusers and 11 users of OC. Net endothelial release rates of t-PA were calculated as the product of the arteriovenous concentration gradient and forearm plasma flow in response to intra-arterial bradykinin (BK: 12.5–50 ng · 100 ml tissue−1· min−1) and sodium nitroprusside (SNP: 1.0–4.0 μg · 100 ml tissue−1· min−1). Net release of t-PA antigen and increment in t-PA activity across the forearm to BK increased ( P < 0.01) in a dose-dependent fashion and to similar extents in the nonusers and users of OC. At the highest BK dose, net release of t-PA antigen was 64.5 ± 8.2 and 66.2 ± 15.4 ng · 100 ml tissue−1· min−1in the nonusers and users of OC, whereas the net increment in t-PA activity was 18.6 ± 3.0 and 16.0 ± 2.0 IU · 100 ml tissue−1· min−1, respectively. There was no effect of SNP on t-PA release in either group. These results indicate that endothelial t-PA release is not altered in premenopausal women who use oral contraception.
Interrelationships of serum testosterone and free testosterone index with FFM and strength in aging men
Authors: Roy Tracey Ann , Blackman Marc R. , Harman S. Mitchell , Tobin Jordan D. , Schrager Matthew , Metter E. Jeffery
Published: February 2015

Muscle mass and strength losses during aging may be associated with declining levels of serum testosterone (T) in men. Few studies have shown a direct relationship between T and muscle mass and strength. Subjects were 262 men, aged 24–90 yr, from the Baltimore Longitudinal Study of Aging, who had T and sex hormone-binding globulin sex hormone-binding globulin (SHBG) measurements, from which the free T index (FTI) was calculated (T/SHBG) from serum samples collected longitudinally since 1963, total body fat mass and arm and leg fat-free mass (FFM) by dual-energy X-ray absorptiometry and arm and leg strength by dynanomometry. Mixed-effects models estimated T and FTI at the time of mass and strength measurements. Age, total body fat, arm and leg FFM, T, and FTI were significantly associated with concentric and eccentric strength. FTI, not T, was modestly, but directly, related to arm and leg strength after fat, arm and leg FFM, height, and age were accounted for and indirectly through body mass. FTI is a better predictor of arm and leg strength than T in aging men.
Human protein metabolism: its measurement and regulation
Authors: Liu Zhenqi , Barrett Eugene J.
Published: February 2015

The body's protein mass not only provides architectural support for cells but also serves vital roles in maintaining their function and survival. The whole body protein pool, as well as that of individual tissues, is determined by the balance between the processes of protein synthesis and degradation. These in turn are regulated by interactions among hormonal, nutritional, neural, inflammatory, and other influences. Prolonged changes in either the synthetic or degradative processes (or both) that cause protein wasting increase morbidity and mortality. The application of tracer kinetic methods, combined with measurements of the activity of components of the cellular signaling pathways involved in protein synthesis and degradation, affords new insights into the regulation of both protein synthesis and breakdown in vivo. These insights, including those from studies of insulin, insulin-like growth factor I, growth hormone, and amino acid-mediated regulation of muscle and whole body protein turnover, provide opportunities to develop and test therapeutic approaches with promise to minimize or prevent these adverse health consequences.
Insulin-stimulated cytosol alkalinization facilitates optimal activation of glucose transport in cardiomyocytes
Authors: Yang Jing , Gillingham Alison K. , Hodel Alois , Koumanov Françoise , Woodward Brian , Holman Geoffrey D.
Published: February 2015

Abnormalities in intracellular pH regulation have been proposed to be important in type 2 diabetes and the associated cardiomyopathy and hypertension. We have therefore investigated the dependence of insulin-stimulated glucose transport on cytosolic pH in cardiomyocytes. Insulin treatment of cardiomyocytes resulted in a marked alkalinization of the cytoplasm as measured using carboxy-semi-napthorhodofluor-1. The alkalinizing effect of insulin was blocked by treatment with either cariporide (which inhibits the Na+/H+exchanger) or by bafilomycin A1 (which inhibits H+-ATPase activity). After treatments with cariporide or bafilomycin A1, insulin stimulation of insulin receptor and insulin receptor substrate-1 phosphorylation and Akt activity were normal. In contrast, glucose transport activity and the levels of functional GLUT4 at the plasma membrane (detected using an exofacial photolabel) were reduced by ∼50%. Immunocytochemical analysis revealed that insulin treatment caused a translocation of the GLUT4 from perinuclear structures and increased its co-localization with cell surface syntaxin 4. However, neither cariporide nor bafilomycin A1 treatment reduced the translocation of immunodetectable GLUT4 to the sarcolemma region of the cell. It is therefore hypothesized that insulin-stimulated cytosol alkalinization facilitates the final stages of translocation and incorporation of fully functional GLUT4 at the surface-limiting membrane.
Lipid metabolism and resistin gene expression in insulin-resistant Fischer 344 rats
Authors: Levy James R. , Davenport Byrd , Clore John N. , Stevens Wayne
Published: February 2015

The interrelationship between insulin and leptin resistance in young Fischer 344 (F344) rats was studied. Young F344 and Sprague-Dawley (SD) rats were fed regular chow. F344 animals had two- to threefold higher insulin and triglyceride concentrations and increased stores of triglycerides within liver and muscle. F344 animals gained more body fat. Both acyl-CoA oxidase (ACO) and carnitine palmitoyltransferase I gene expression were 20–50% less in F344 animals than in age-matched SD animals. Peroxisome proliferator-activated receptor-α gene expression was reduced in 70-day-old F344 animals. Finally, resistin gene expression was similar in 70-day-old SD and F344 animals. Resistin gene expression increased fivefold in F344 animals and twofold in SD animals from 70 to 130 days, without a change in insulin sensitivity. We conclude that young F344 animals have both insulin and leptin resistance, which may lead to diminished fatty oxidation and accumulation of triglycerides in insulin-sensitive target tissues. We did not detect a role for resistin in the etiology of insulin resistance in F344 animals.
The other side of the orexins: endocrine and metabolic actions
Authors: Taylor Meghan M. , Samson Willis K.
Published: February 2015

Although it is clear that the orexin/hypocretin peptides have a significant, physiologically relevant role in sleep/wakefulness, a broader picture has emerged indicating metabolic actions that may depend upon both neural and endocrine mechanisms for their manifestation. The ability of exogenous peptide to activate sympathetic tone, increase locomotor activity, and alter feeding behavior, together with the observed alterations in those functions in knockout animals, strongly suggests important neural actions of the endogenous orexins/hypocretins. Likewise, the action of exogenously administered peptides to alter endocrine function, in particular corticotropin release, has now been mirrored by potential endocrinopathies in knockout animals. Thus these pluripotent peptides hold great potential not only for the treatment of human narcolepsy but also to provide insight into the coordinated regulation of multiple physiological systems.
Anabolic action of insulin on skin wound protein is augmented by exogenous amino acids
Authors: Zhang Xiao-Jun , Chinkes David L. , Irtun Øivind , Wolfe Robert R.
Published: February 2015

To investigate the metabolic basis of skin wound healing, we measured in anesthetized rabbits the responses of protein kinetics in scalded skin to insulin and amino acids.l-[ ring-13C6]Phe was infused on the 7th day after the ear was scalded, and the scalded ear was used as an arteriovenous unit to reflect protein kinetics in skin wound. The ipsilateral carotid artery was clamped to control the wound blood flow within four- to fivefold the normal skin rate to measure the enrichment difference in the scalded ear during hyperaminoacidemia. Neither insulin (2.5 mU · kg−1· min−1) nor amino acid (2.5 mg · kg−1· min−1) infusion alone improved net protein balance in the skin wound. In contrast, combined infusion of insulin and amino acids increased the net protein balance in skin wound from −6.5 ± 4.5 to 1.4 ± 5.2 μmol · 100 g−1· h−1( P < 0.01, control vs. insulin plus amino acids). We conclude that there is an interactive effect of insulin and sufficient amino acid supply on protein metabolism in skin wound, meaning that their combined anabolic effect is greater than the sum of their individual effects.
Gender difference in the Oatp1-mediated tubular reabsorption of estradiol 17β-d-glucuronide in rats
Authors: Gotoh Yasumasa , Kato Yukio , Stieger Bruno , Meier Peter J. , Sugiyama Yuichi
Published: February 2015

The gender difference in the urinary excretion of estradiol-17β-glucuronide (E2-17βG) was examined in rats. The urinary clearance of E2-17βG was >250 times lower in male than in female rats. No such major gender difference was observed in its biliary excretion or metabolism in kidney homogenate. Both plasma protein binding and inulin clearance were comparable in male and female rats, suggesting that this gender difference cannot be explained by glomerular filtration. The urinary clearance with respect to the plasma unbound E2-17βG in male rats was <1% of the glomerular filtration rate, indicating its potential reabsorption by the kidney, and this increased to a level comparable with that found in female rats when dibromosulfophthalein was coinfused. A marked increase in E2-17βG urinary excretion was also observed in male rats that had undergone orchidectomy. Testosterone injections given to female rats reduced the urinary excretion to a level comparable with that of control male rats. The concomitant change in the expression of the gene product for organic anion-transporting polypeptide Oatp1, of which E2-17βG is a typical substrate, was found in the kidney membrane fractions after these treatments. These results suggest that urinary E2-17βG excretion is subject to hormonal regulation and that the large gender difference can be explained by regulation in Oatp1-mediated reabsorption.
Peptide transport in the mammary gland: expression and distribution of PEPT2 mRNA and protein
Authors: Groneberg David A. , Döring Frank , Theis Stephan , Nickolaus Monika , Fischer Axel , Daniel Hannelore
Published: February 2015

The lactating mammary gland utilizes free plasma amino acids as well as those derived by hydrolysis from circulating short-chain peptides for protein synthesis. Apart from the major route of amino acid nitrogen delivery to the gland by the various transporters for free amino acids, it has been suggested that dipeptides may also be taken up in intact form to serve as a source of amino acids. The identification of peptide transporters in the mammary gland may therefore provide new insights into protein metabolism and secretion by the gland. The expression and distribution of the high-affinity type proton-coupled peptide transporter PEPT2 were investigated in rat lactating mammary gland as well as in human epithelial cells derived from breast milk. By use of RT-PCR, PEPT2 mRNA was detected in rat mammary gland extracts and human milk epithelial cells. The expression pattern of PEPT2 mRNA revealed a localization in epithelial cells of ducts and glands by nonisotopic high resolution in situ hybridization. In addition, immunohistochemistry was carried out and showed transporter immunoreactivity in the same epithelial cells of the glands and ducts. In addition, two-electrode voltage clamp recordings using PEPT2-expressing Xenopus laevis oocytes demonstrated positive inward currents induced by selected dipeptides that may play a role in aminonitrogen handling in mammalian mammary gland. Taken together, these data suggest that PEPT2 is expressed in mammary gland epithelia, in which it may contribute to the reuptake of short-chain peptides derived from hydrolysis of milk proteins secreted into the lumen. Whereas PEPT2 also transports a variety of drugs, such as selected β-lactams, angiotensin-converting enzyme inhibitors, and antiviral and anticancer metabolites, their efficient reabsorption via PEPT2 may reduce the burden of xenobiotics in milk.
Oxytocin stimulation of RGS2 mRNA expression in cultured human myometrial cells
Authors: Park Eun Sung , Echetebu Clement O. , Soloff Solweig , Soloff Melvyn S.
Published: February 2015

Regulators of G protein signaling (RGS proteins) interact with Gαq and Gαi and accelerate GTPase activity. These proteins have been characterized only within the past few years, so our understanding of their importance is still preliminary. We examined the effect of oxytocin on RGS2 mRNA expression to help determine the role of RGS proteins in oxytocin signaling in human myometrial cells in primary culture. Oxytocin increased RGS2 mRNA concentration maximally by 1 or 2 h in a dose-dependent and agonist-specific manner. RGS2 mRNA levels were also elevated by treatment with Ca2+ ionophore, phorbol ester, or forskolin. Oxytocin's effects were completely inhibited by an intracellular Ca2+ chelator and partially blocked by a protein kinase C inhibitor, indicating that intracellular Ca2+ concentration is the primary signal for oxytocin elevation of RGS2 mRNA levels. Use of pharmacological inhibitors indicated that part of oxytocin-stimulated RGS2 mRNA expression is mediated by Gi/tyrosine kinase activities. Although oxytocin does not stimulate increases in intracellular cAMP concentration, agents that elevate intracellular cAMP concentrations and cause myometrial relaxation may possibly cause heterologous desensitization to oxytocin via RGS2 expression. These results suggest that RGS2 may be important in regulating the myometrial response to oxytocin.
Calcitonin gene expression induced by lipopolysaccharide in the rat pituitary
Authors: Kiriyama Yoshimitsu , Nomura Yasuyuki , Tokumitsu Yukiko
Published: February 2015

Procalcitonin (PCT), the precursor protein of calcitonin (CT), has been considered recently as a significant indicator of bacterial infection and sepsis. However, the major source of PCT in sepsis remains unclear. The hypothalamic-pituitary-adrenal axis is activated during sepsis. Moreover, immunoreactive CT (iCT) can be detected in the pituitary. Therefore, we examined the effects of lipopolysaccharide (LPS) administration on CT mRNA expression in the pituitary. After administration of LPS, CT mRNA expression in the pituitary was increased significantly. The increase of CT mRNA was associated with significant elevations of the iCT levels in the serum. These results imply that the pituitary is one of the sources of the serum PCT during sepsis.
Dual actions of lanthanides on ACTH-inhibited leak K+channels
Authors: Enyeart John J. , Xu Lin , Enyeart Judith A.
Published: February 2015

Bovine adrenal zona fasciculata cells express background K+channels ( IACchannels) whose activity is potently inhibited by ACTH. In whole cell patch clamp recordings, it was discovered that the trivalent lanthanides (Ln3+s) lanthanum and ytterbium interact with two binding sites to modulate K+flow through these channels. Despite large differences in ionic radii, these Ln3+s inhibited IACchannels half-maximally with IC50values near 50 μM. In addition, these Ln3+s blocked and reversed ACTH-mediated inhibition of IACK+channels at similar concentrations. The Ln3+s did not alter inhibition of IACby angiotensin II or cAMP. Ln3+-induced uncoupling of ACTH receptor activation from IACinhibition was prevented by raising the external Ca2+concentration from 2 to 10 mM. The divalent cation Ni2+(500 μM) also blocked ACTH-dependent inhibition of IACthrough a Ca2+-sensitive mechanism. The results are consistent with a model in which Ln3+s produce opposing actions on IACK+currents through two separate binding sites. In addition to directly inhibiting IAC, Ln3+s (and Ni2+) bind with high affinity to a Ca2+-selective site associated with the ACTH receptor. By displacing Ca2+from this site, Ln3+s prevent ACTH from binding and accelerate its dissociation. These results identify Ln3+s as a relatively potent group of noncompetitive ACTH receptor antagonists. Allosteric actions of trivalent and divalent metal cations on hormone binding, mediated through Ca2+-specific sites, may be common to a variety of peptide hormone receptors.
Urocortin: a mechanism for the sustained activation of the HPA axis in the late-gestation ovine fetus?
Authors: Holloway Alison C. , Howe David C. , Chan Gabriel , Clifton Vicki L. , Smith Roger , Challis John R. G.
Published: February 2015

We hypothesized that urocortin might be produced in the pituitary of the late-gestation ovine fetus in a manner that could contribute to the regulation of ACTH output. We used in situ hybridization and immunohistochemistry to identify urocortin mRNA and protein in late-gestation fetal pituitary tissue. Levels of urocortin mRNA rose during late gestation and were associated temporally with rising concentrations of pituitary proopiomelanocortin (POMC) mRNA. Urocortin was localized both to cells expressing ACTH and to non-ACTH cells by use of dual immunofluorescence histochemistry. Transfection of pituitary cultures with urocortin antisense probe reduced ACTH output, whereas added urocortin stimulated ACTH output from cultured pituitary cells. Cortisol infusion for 96 h in chronically catheterized late-gestation fetal sheep significantly stimulated levels of pituitary urocortin mRNA. We conclude that urocortin is expressed in the ovine fetal pituitary and localizes with, and can stimulate output of, ACTH. Regulation of urocortin by cortisol suggests a mechanism to override negative feedback and sustain feedforward of fetal hypothalamic-pituitary-adrenal function, leading to birth.