bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional alteration of dihydropyridine-sensitive Ca2+channels in the adrenal glomerulosa of pregnant rats
Authors: Simaan May , Picard Serge , St-Louis Jean , Brochu Michèle
Published: December 2017

Our previous work on aldosterone secretion suggested that dihydropyridine-sensitive calcium channels, one type of voltage-dependent calcium channels (VDCC), are functionally impaired in adrenal capsule preparations from the pregnant rat. The aim of this study was to determine whether, during pregnancy, the density and/or activity of these channels is altered in the adrenal zona glomerulosa. These VDCC measured with [3H]nitrendipine binding were not different between membrane preparations of nonpregnant and pregnant rats. Western blots were performed using two different antibodies, a polyclonal (PcAb) directed against the α1-subunit of VDCC and a monoclonal (McAb) that recognizes an intracellular domain of that protein. McAb immunoreactivity showed a significant decrease in preparations from pregnant rats, whereas no difference was observed with PcAb. VDCC activity was estimated by45Ca2+uptake in isolated adrenal cortex and by intracellular calcium concentration ([Ca2+]i) in adrenal glomerulosa cells with the Ca2+probe fura PE3. These measurements revealed that KCl stimulation produced greater Ca2+influx in nonpregnant than in pregnant rats. Nifedipine (a blocker of VDCC) inhibited this stimulation only in nonpregnant rats, whereas BAY K 8644 (an activator of VDCC) increased Ca2+influx in pregnant rats only. These data suggest that, during pregnancy, the altered regulation of calcium homeostasis in adrenal glomerulosa is linked to a conformational alteration of VDCC.
GLUT-1 reduces hypoxia-induced apoptosis and JNK pathway activation
Authors: Lin Zhiwu , Weinberg Joel M. , Malhotra Ricky , Merritt Steven E. , Holzman Lawrence B. , Brosius Frank C.
Published: December 2017

Many studies have suggested that enhanced glucose uptake protects cells from hypoxic injury. More recently, it has become clear that hypoxia induces apoptosis as well as necrotic cell death. We have previously shown that hypoxia-induced apoptosis can be prevented by glucose uptake and glycolytic metabolism in cardiac myocytes. To test whether increasing the number of glucose transporters on the plasma membrane of cells could elicit a similar protective response, independent of the levels of extracellular glucose, we overexpressed the facilitative glucose transporter GLUT-1 in a vascular smooth muscle cell line. After 4 h of hypoxia, the percentage of cells that showed morphological changes of apoptosis was 30.5 ± 2.6% in control cells and only 6.0 ± 1.1 and 3.9 ± 0.3% in GLUT-1-overexpressing cells. Similar protection against cell death and apoptosis was seen in GLUT-1-overexpressing cells treated for 6 h with the electron transport inhibitor rotenone. In addition, hypoxia and rotenone stimulated c-Jun-NH2-terminal kinase (JNK) activity >10-fold in control cell lines, and this activation was markedly reduced in GLUT-1-overexpressing cell lines. A catalytically inactive mutant of MEKK1, an upstream kinase in the JNK pathway, reduced hypoxia-induced apoptosis by 39%. These findings show that GLUT-1 overexpression prevents hypoxia-induced apoptosis possibly via inhibition of stress-activated protein kinase pathway activation.
Somatostatin restrains the secretion of glucagon-like peptide-1 and -2 from isolated perfused porcine ileum
Authors: Hansen Lene , Hartmann Bolette , Bisgaard Thue , Mineo Hitoshi , Jørgensen Peer N. , Holst Jens J.
Published: December 2017

Suspecting that paracrine inhibition might influence neuronal regulation of the endocrine L cells, we studied the role of somatostatin (SS) in the regulation of the secretion of the proglucagon-derived hormones glucagon-like peptide-1 and -2 (GLP-1 and GLP-2). This was examined using the isolated perfused porcine ileum stimulated with acetylcholine (ACh, 10−6M), neuromedin C (NC, 10−8M), and electrical nerve stimulation (NS) with or without α-adrenergic blockade (phentolamine 10−5M), and perfusion with a high-affinity monoclonal antibody against SS. ACh and NC significantly increased GLP secretion, whereas NS had little effect. SS immunoneutralization increased GLP secretion eight- to ninefold but had little influence on the GLP responses to ACh, NC, and NS. Basal SS secretion (mainly SS28) was unaffected by NS alone. Phentolamine + NS and NC abstract strongly stimulated release mainly of SS14, whereas ACh had little effect. Infused intravascularly, SS14 weakly and SS28 strongly inhibited GLP secretion. We conclude that GLP secretion is tonically inhibited by a local release of SS28 from epithelial paracrine cells, whereas SS14, supposedly derived from enteric neurons, only weakly influences GLP secretion.
Insulin-mediated translocation of GLUT-4-containing vesicles is preserved in denervated muscles
Authors: Zhou Min , Vallega Gino , Kandror Konstantin V. , Pilch Paul F.
Published: December 2017

Skeletal muscle denervation decreases insulin-sensitive glucose uptake into this tissue as a result of marked GLUT-4 protein downregulation (∼20% of controls). The process of insulin-stimulated glucose transport in muscle requires the movement or translocation of intracellular GLUT-4-rich vesicles to the cell surface, and it is accompanied by the translocation of several additional vesicular cargo proteins. Thus examining GLUT-4 translocation in muscles from denervated animals allows us to determine whether the loss of a major cargo protein, GLUT-4, affects the insulin-dependent behavior of the remaining cargo proteins. We find no difference, control vs. denervated, in the insulin-dependent translocation of the insulin-responsive aminopeptidase (IRAP) and the receptors for transferrin and insulin-like growth factor II/mannose 6-phosphate, proteins that completely (IRAP) or partially co-localize with GLUT-4. We conclude that 1) denervation of skeletal muscle does not block the specific branch of insulin signaling pathway that connects receptor proximal events to intracellular GLUT-4-vesicles, and 2) normal levels of GLUT-4 protein are not necessary for the structural organization and insulin-sensitive translocation of its cognate intracellular compartment. Muscle denervation also causes a twofold increase in GLUT-1. In normal muscle, all GLUT-1 is present at the cell surface, but in denervated muscle a significant fraction (25.1 ± 6.1%) of this transporter is found in intracellular vesicles that have the same sedimentation coefficient as GLUT-4-containing vesicles but can be separated from the latter by immunoadsorption. These GLUT-1-containing vesicles respond to insulin and translocate to the cell surface. Thus the formation of insulin-sensitive GLUT-1-containing vesicles in denervated muscle may be a compensatory mechanism for the decreased level of GLUT-4.
Peroxynitrite production by TNF-α and IL-1β: implication for suppression of osteoblastic differentiation
Authors: Hikiji Hisako , Shin Wee Soo , Koizumi Toshiyuki , Takato Tsuyoshi , Susami Takafumi , Koizumi Yoko , Okai-Matsuo Yoko , Toyo-Oka Teruhiko
Published: December 2017

To determine the roles of nitric oxide (NO) and its metabolite, peroxynitrite (ONOO−), on osteoblastic activation, we investigated the effects of a NO donor [ethanamine, 2,2′-(hydroxynitrosohydrazono)bis- (dNO)], an [Formula: see text] donor (pyrogallol), and an ONOO− scavenger (urate) on alkaline phosphatase (ALPase) activity and osteocalcin gene expression, which are indexes of osteoblastic differentiation. dNO elevated ALPase activity in the osteogenic MC3T3-E1 cell line. The combination of dNO and pyrogallol reduced both ALPase activity and osteocalcin gene expression. Because both indexes were recovered by urate, ONOO−, unlike NO itself, inhibited the osteoblastic differentiation. Furthermore, treatment with a combination of the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) was found to yield ONOO−as well as NO and [Formula: see text]. The reductions in ALPase activity and osteocalcin gene expression were also restored by urate. We conclude that ONOO− produced by TNF-α and IL-1β, but not NO per se, would overcome the stimulatory effect of NO on osteoblastic activity and inhibit osteoblastic differentiation.
Short-term effects of growth hormone on myocardial glucose uptake in healthy humans
Authors: Bøtker Hans Erik , Wiggers Henrik , Bøttcher Morten , Christiansen Jens Sandahl , Nielsen Torsten Toftegaard , Gjedde Albert , Schmitz Ole
Published: December 2017

Cardiac muscle is characterized by insulin resistance in specific heart diseases such as coronary artery disease and congestive heart failure, but not in generalized disorders like diabetes mellitus and essential hypertension when cardiac manifestations are absent. To examine whether the insulin antagonistic effect of growth hormone (GH) acts upon the heart, we compared insulin-stimulated whole body and myocardial glucose uptake with and without GH administration during a 3.5-h euglycemic-hyperinsulinemic clamp in eight healthy males. Myocardial 2-deoxy-2-[18F]fluoro-d-glucose uptake was measured with positron emission tomography. The data were converted to myocardial glucose uptake by tracer kinetic analysis. GH did not change the rate-pressure product. GH decreased whole body insulin-stimulated glucose disposal by 26% (48.0 ± 12.1 vs. control 62.8 ± 6.1 μmol ⋅ kg−1⋅ min−1, P < 0.02). Free fatty acids were suppressed to a similar extent with and without GH during the insulin clamp. Insulin-stimulated myocardial glucose uptake was similar in the presence and in the absence of GH (0.34 ± 0.05 and 0.31 ± 0.03 μmol ⋅ g−1⋅ min−1, P = 0.18). In conclusion, GH does not impair insulin-stimulated myocardial glucose uptake despite a considerable whole body insulin antagonistic effect. Myocardial insulin resistance is not an inherent consequence of whole body insulin resistance.
Somatotropin increases protein balance independent of insulin's effects on protein metabolism in growing pigs
Authors: Vann Rhonda C. , Nguyen Hanh V. , Reeds Peter J. , Steele Norman C. , Deaver Daniel R. , Davis Teresa A.
Published: December 2017

Somatotropin (ST) administration enhances protein deposition and elicits profound metabolic responses, including hyperinsulinemia. To determine whether the anabolic effect of ST is due to hyperinsulinemia, pair-fed weight-matched growing swine were treated with porcine ST (150 μg · kg body wt−1 · day−1) or diluent for 7 days ( n = 6/group, ∼20 kg). Then pancreatic glucose-amino acid clamps were performed after an overnight fast. The objective was to reproduce the insulin levels of 1) fasted control and ST pigs (basal insulin, 5 μU/ml), 2) fed control pigs (low insulin, 20 μU/ml), and 3) fed ST pigs (high insulin, 50 μU/ml). Amino acid and glucose disposal rates were determined from the infusion rates necessary to maintain preclamp blood levels of these substrates. Whole body nonoxidative leucine disposal (NOLD), leucine appearance (Ra), and leucine oxidation were determined with primed, continuous infusions of [13C]leucine and [14C]bicarbonate. ST treatment was associated with higher NOLD and protein balance and lower leucine oxidation and amino acid and glucose disposals. Insulin lowered Ra and increased leucine oxidation, protein balance, and amino acid and glucose disposals. These effects of insulin were suppressed by ST treatment; however, the protein balance remained higher in ST pigs. The results show that ST treatment inhibits insulin's effects on protein metabolism and indicate that the stimulation of protein deposition by ST treatment is not mediated by insulin. Comparison of the protein metabolic responses to ST treatment during the basal fasting period with those in the fully fed state from a previous study suggests that the mechanism by which ST treatment enhances protein deposition is influenced by feeding status.
Biological activities of recombinant chicken leptin C4S analog compared with unmodified leptins
Authors: Dridi S. , Raver N. , Gussakovsky E. E. , Derouet M. , Picard M. , Gertler A. , Taouis M.
Published: December 2017

The chicken leptin sequence, in contrast to mammalian leptins, contains an unpaired Cys at position 3 of the original cDNA ( AF012727 ). The presence of an extra Cys may confer a different structure and affect the leptin's biological activity. To address this, we studied the effects of wild-type and mutated (C4S) chicken leptins in vitro and in vivo and compared them with mammalian leptin prepared from ovine leptin cDNA. The prokaryotic expression vector pMON, encoding full-size A(−1) chicken leptin ( AF012727 ), was mutated using a mutagenesis kit, yielding the C4S analog. Escherichia coli cells transformed with this vector overexpressed large amounts of chicken leptin C4S upon induction with nalidixic acid. The expressed protein, found in the inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding three electrophoretically pure fractions, eluted from the column by 100, 125, and 150 mM NaCl, respectively. All three fractions showed a single band of the expected molecular mass (16 kDa) and were composed of >95% monomeric protein. Proper refolding was evidenced by comparing the circular dichroism spectrum of the analog with spectra of nonmutated chicken and ovine leptins. The biological activity of the C4S analog was evidenced by its ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin receptor construct similar to its nonmutated counterpart, indicating that Cys4 plays no role in leptin activity. The in vitro activity of both wild-type and mutated chicken leptins was ∼10-fold lower than that of ovine leptin. After intravenous or intraperitoneal injections, C4S analog and the nonmutated chicken and ovine leptins all lowered the food intake of starved 9-day-old broiler or 5-wk-old layer male chickens by 11–34%. Monitoring food behavior revealed that the attenuated food intake resulted not from a decreased number of approaches to the feeders but from a decrease in the average time spent eating during each approach.
Aldosterone- and testosterone-mediated intracellular calcium response in skeletal muscle cell cultures
Authors: Estrada Manuel , Liberona José Luis , Miranda Manuel , Jaimovich Enrique
Published: December 2017

Fast nongenomic steroid actions in several cell types seem to be mediated by second messengers such as intracellular calcium ([Ca2+]i) and inositol 1,4,5-trisphosphate (IP3). We have shown the presence of both slow calcium transients and IP3receptors associated with cell nuclei in cultured skeletal muscle cells. The effect of steroids on [Ca2+]iwas monitored in Fluo 3-acetoxymethyl ester-loaded myotubes by either confocal microscopy or fluorescence microscopy, with the use of out-of-focus fluorescence elimination. The mass of IP3was determined by radioreceptor displacement assay. [Ca2+]ichanges after either aldosterone (10–100 nM) or testosterone (50–100 nM) were observed; a relatively fast (<2 min) calcium transient, frequently accompanied by oscillations, was evident with both hormones. A slow rise in [Ca2+]ithat reached its maximum after a 30-min exposure to aldosterone was also observed. Calcium responses seem to be fairly specific for aldosterone and testosterone, because several other steroid hormones do not induce detectable changes in fluorescence, even at 100-fold higher concentrations. The mass of IP3increased transiently to reach two- to threefold the basal level 45 s after addition of either aldosterone or testosterone, and the IP3transient was more rapid than the fast calcium signal. Spironolactone, an inhibitor of the intracellular aldosterone receptor, or cyproterone acetate, an inhibitor of the testosterone receptor, had no effect on the fast [Ca2+]isignal or in the increase in IP3mass. These signals could mean that there are distinct nongenomic pathways for the action of these two steroids in skeletal muscle cells.
Inhibitory effect of NF-κB on 1,25-dihydroxyvitamin D3and retinoid X receptor function
Authors: Farmer Paul K. , He Xiaofei , Schmitz M. Lienhard , Rubin Janet , Nanes Mark S.
Published: December 2017

Responsiveness to 1,25-dihydroxyvitamin D3[1,25(OH)2D3] may be diminished in osteoporosis and inflammatory arthritis. The inflammatory cytokine tumor necrosis factor-α (TNF-α) is produced in excess in these disorders and has been shown to decrease osteoblast transcriptional responsiveness to vitamin D and to inhibit the binding of the vitamin D receptor (VDR) and its nuclear partner the retinoid X receptor (RXR) to DNA. Previous studies have shown that a vitamin D (VDRE) or retinoid X DNA response element (RXRE) is sufficient to confer TNF-α inhibition of vitamin D or retinoid-stimulated transcription in the absence of known TNF-α-responsive DNA sequences. We tested the hypothesis that the TNF-α-stimulated transcription factor nuclear factor (NF)-κB could, in part, mediate TNF-α action by inhibiting the transcriptional potency of the VDR and RXR at their cognate cis regulatory sites. Osteoblastic ROS 17/2.8 cells transfected with a dose of NF-κB comparable to that stimulated by TNF-α decreased 1,25(OH)2D3-stimulated transcription. This inhibitory effect of NF-κB was not observed on basal transcription of a heterologous reporter in the absence of the VDRE. The effects of NF-κB and TNF-α were comparable but not additive. COS-7 cells were cotransfected with reporters under the regulation of VDRE or RXRE along with vectors expressing VDR, RXR, and NF-κB nuclear proteins. Reconstituted NF-κB and the NF-κB subunit p65 alone, but not p50, dose dependently suppressed basal and ligand-stimulated transcription. p65 overexpression completely abrogated enhanced VDRE-mediated transcriptional activity in response to 1,25(OH)2D3. Electrophoretic mobility shift experiments did not reveal a direct effect of recombinant NF-κB or its individual subunits on the binding of heterodimeric VDR-RXR to DNA. These results suggest that TNF-α inhibition of hormone-stimulated transcriptional activation may be mediated by activation of NF-κB. In contrast, the inhibitory effect of TNF-α on binding of receptors to DNA is unlikely to be mediated by NF-κB and is not necessary for inhibition of transcription.
Direct measurement of the lumped constant for 2-deoxy-[1-14C]glucose in vivo in human skeletal muscle
Authors: Utriainen Tapio , Lovisatti Stefania , Mäkimattila Sari , Bertoldo Alessandra , Weintraub Susan , DeFronzo Ralph , Cobelli Claudio , Yki-Järvinen Hannele
Published: December 2017

The lumped constant (LC) is used to convert the clearance rate of 2-deoxy-d-glucose (2-DGcr) to that of glucose (Glccr). There are currently no data to validate the widely used assumption of an LC of 1.0 for human skeletal muscle. We determined the LC for 2-deoxy-[1-14C]glucose (2-DG) in 18 normal male subjects (age, 29 ± 2 yr; body mass index, 24.8 ± 0.8 kg/m2) after an overnight fast and during physiological (1 mU · kg−1· min−1insulin infusion for 180 min) and supraphysiological (5 mU · kg−1· min−1insulin infusion for 180 min) hyperinsulinemic conditions. Normoglycemia was maintained with the euglycemic clamp technique. The LC was measured directly with the use of a novel triple tracer-based method. [3-3H]glucose, 2-[1-14C]DG, and [12C]mannitol (Man) were injected as a bolus into the brachial artery. The concentrations of [3-3H]glucose and 2-[1-14C]DG (dpm/ml plasma) and of Man (μmol/l) were determined in 50 blood samples withdrawn from the ipsilateral deep forearm vein over 15 min after the bolus injection. The LC was calculated by a formula involving blood flow calculated from Man and the Glccrand 2-DGcr. The LC averaged 1.26 ± 0.08 (range 1.06–1.43), 1.15 ± 0.05 (0.99–1.39), and 1.18 ± 0.05 (0.97–1.37) under fasting conditions and during the 1 and 5 mU · kg−1· min−1insulin infusions (not significant between the different insulin concentrations, mean LC = 1.2, P < 0.01 vs. 1.0). We conclude that, in normal subjects, the LC for 2-DG in human skeletal muscle is constant over a wide range of insulin concentrations and averages 1.2.
Parathyroid hormone-related protein induces G1 phase growth arrest of vascular smooth muscle cells
Authors: Stuart William D. , Maeda Shigeto , Khera Paramjit , Fagin James A. , Clemens Thomas L.
Published: December 2017

In this study, we investigated the mechanisms responsible for the growth-inhibitory action of parathyroid hormone-related protein (PTHRP) in A10 vascular smooth muscle cells (VSMC). Fluorescence-activated cell sorting analysis of serum-stimulated VSMC treated with PTHRP or dibutyryl-cAMP (DBcAMP) demonstrated an enrichment of cells in G1 and a reduction in the S phase. Measurement of DNA synthesis in platelet-derived growth factor-stimulated VSMC treated with DBcAMP revealed that cells became refractory to growth inhibition by 12–16 h, consistent with blockade in mid-G1. cAMP treatment blunted the serum-induced rise in cyclin D1 during cell cycle progression without altering levels of the cyclin-dependent kinase cdk4 or cyclin E and its associated kinase, cdk2. Exposure of cells to PTHRP or cAMP resulted in a reduction in retinoblastoma gene product (Rb) phosphorylation. Immunoblotting of extracts from cAMP-treated cells with antibodies to cdk inhibitors revealed a striking increase in p27kip1abundance coincident with the G1 block. Immunoprecipitation with an anti-cyclin D1 antibody of cell lysates prepared from cAMP-treated cells followed by immunoblotting with antisera to p27kip1disclosed a threefold increase in p27kip1associated with cyclin D1 compared with lysates treated with serum alone. We conclude that PTHRP, by increasing intracellular cAMP, induces VSMC cycle arrest in mid-G1. This occurs secondary to a suppression in cyclin D1 and induction of p27kip1expression, which in turn inhibits Rb phosphorylation.
Enhanced intestinal synthesis of polyamines from proline in cortisol-treated piglets
Authors: Wu Guoyao , Flynn Nick E. , Knabe Darrell A.
Published: December 2017

This study was conducted to determine a role for cortisol in regulating intestinal ornithine decarboxylase (ODC) activity and to identify the metabolic sources of ornithine for intestinal polyamine synthesis in suckling pigs. Thirty-two 21-day-old suckling pigs were randomly assigned to one of four groups with eight animals each and received daily intramuscular injections of vehicle solution (sesame oil; control), hydrocortisone 21-acetate (HYD; 25 mg/kg body wt), RU-486 (10 mg/kg body wt, a potent blocker of glucocorticoid receptors), or HYD plus RU-486 for two consecutive days. At 29 days of age, pigs were killed for preparation of jejunal enterocytes. The cytosolic fraction was prepared for determining ODC activity. For metabolic studies, enterocytes were incubated for 45 min at 37°C in 2 ml of Krebs-bicarbonate buffer (pH 7.4) containing 1 mM [U-14C]arginine, 1 mM [U-14C]ornithine, 1 mM [U-14C]glutamine, or 1 mM [U-14C]proline plus 1 mM glutamine. Cortisol administration increased intestinal ODC activity by 230%, polyamine (putrescine, spermidine, and spermine) synthesis from ornithine and proline by 75–180%, and intracellular polyamine concentrations by 45–83%. Polyamine synthesis from arginine was not detected in enterocytes of control pigs but was induced in cells of cortisol-treated pigs. There was no detectable synthesis of polyamines from glutamine in enterocytes of all groups of pigs. The stimulating effects of cortisol on intestinal ODC activity and polyamine synthesis were abolished by coadministration of RU-486. Our data indicate that an increase in plasma cortisol concentrations stimulates intestinal polyamine synthesis via a glucocorticoid receptor-mediated mechanism and that proline (an abundant amino acid in milk) is a major source of ornithine for intestinal polyamine synthesis in suckling neonates.
Regulation of thyroid cell volumes and fluid transport: opposite effects of TSH and iodide on cultured cells
Authors: Cauvi D. , Penel C. , Nlend M. C. , Venot N. , Allasia C. , Chabaud O.
Published: December 2017

Cell volume regulation by thyrotropin (TSH) and iodide, the main effectors involved in thyroid function, was studied in cultured thyroid cells. The mean cell volume, determined by performing 3-D reconstitution on confocal microscopy optical slices from living octadecylrhodamine-labeled cells cultured with both TSH and iodide (control cells), was 3.73 ± 0.06 pl. The absence of iodide resulted in cell hypertrophy (136% of control value) and the absence of TSH in cell shrinkage (81%). These changes mainly affected the cell heights. The effect of TSH on cell volume was mediated by cAMP. The proportion of cytosolic volume (3- O-methyl-d-glucose space vs. total volume) decreased in the absence of iodide (85% of control value) and increased in the absence of TSH (139%), whereas protein content showed the opposite changes (121 and 58%, respectively). The net apical-to-basal fluid transport was also inversely controlled by the two effectors. Iodide thus antagonizes TSH effects on cell volumes and fluid transport, probably via adenylylcyclase downregulation mechanisms. The absence of either iodide or TSH may mimic the imbalance occurring in pathological thyroids.
Transcriptional regulation of connective tissue growth factor by cortisol in osteoblasts
Authors: Pereira Renata C. , Durant Deena , Canalis Ernesto
Published: December 2017

Glucocorticoids have important effects on osteoblastic function. Connective tissue growth factor (CTGF)/insulin-like growth factor binding protein-related protein 2 (IGFBP-rP2) plays a role in cell adhesion and function. We examined the regulation of CTGF/IGFBP-rP2 synthesis in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells). Cortisol caused a time- and dose-dependent increase in CTGF/IGFBP-rP2 mRNA levels in Ob cells. Cycloheximide did not preclude the effect, indicating that it was not protein synthesis dependent. Cortisol increased the rate of CTGF/IGFBP-rP2 transcription and, in transcriptionally arrested Ob cells, did not modify the decay of the transcript. Parathyroid hormone decreased, whereas transforming growth factor-β and, to a lesser extent, bone morphogenetic protein 2 increased CTGF/IGFBP-rP2 mRNA levels, but other hormones and growth factors had no effect. In conclusion, cortisol stimulates CTGF/IGFBP-rP2 transcription in Ob cells. Because CTGF/IGFBP-rP2 binds IGFs, its increased expression could be relevant to the actions of cortisol in bone.
Epidural blockade improves substrate utilization after surgery
Authors: Schricker Thomas , Wykes Linda , Carli Franco
Published: December 2017

The purpose of this study was to test the hypothesis that epidural blockade with local anesthetic improves the anticatabolic effects of glucose after colorectal surgery. Sixteen patients were randomly assigned to undergo a 6-h stable isotope infusion study (3 h fasted, 3 h glucose infusion at 4 mg · kg−1 · min−1) on the second postoperative day with or without perioperative epidural blockade. Protein synthesis, breakdown and oxidation, and glucose production and clearance were assessed byl-[1-13C]leucine and [6,6-2H2]glucose. Epidural blockade did not affect protein and glucose metabolism in the fasted state. Glucose infusion increased glucose clearance ( P < 0.05), accompanied by an increase in the respiratory quotient ( P < 0.05) and a decrease in leucine oxidation ( P < 0.05) only in the presence of epidural blockade. An inverse correlation ( r = −0.74, P< 0.05) between changes in glucose clearance and leucine oxidation was observed. In conclusion, epidural blockade facilitates whole body glucose uptake and inhibits endogenous protein oxidation after abdominal surgery, indicating a shift from a protein to a more glucose-dominated substrate utilization.
Increased expression of GAD65 and GABA in pancreatic β-cells impairs first-phase insulin secretion
Authors: Shi Yuguang , Kanaani Jamil , Menard-Rose Virginie , Ma Yan Hui , Chang Pi-Yun , Hanahan Douglas , Tobin Allan , Grodsky Gerold , Baekkeskov Steinunn
Published: December 2017

The functional role of glutamate decarboxylase (GAD) and its product GABA in pancreatic islets has remained elusive. Mouse β-cells express the larger isoform GAD67, whereas human islets express only the smaller isoform GAD65. We have generated two lines of transgenic mice expressing human GAD65 in pancreatic β-cells (RIP7-hGAD65, Lines 1 and 2) to study the effect that GABA generated by this isoform has on islet cell function. The ascending order of hGAD65 expression and/or activity in β-cells was Line 1 heterozygotes < Line 2 heterozygotes < Line 1 homozygotes. Line 1 heterozygotes have normal glucose tolerance, whereas Line 1 homozygotes and Line 2 heterozygotes exhibit impaired glucose tolerance and inhibition of insulin secretion in vivo in response to glucose. In addition, fasting levels of blood glucose are elevated and insulin is decreased in Line 1 homozygotes. Pancreas perfusion experiments suggest that GABA generated by GAD65 may function as a negative regulator of first-phase insulin secretion in response to glucose by affecting a step proximal to or at the KATP+channel.
Activation of members of the mitogen-activated protein kinase family by glucose in endothelial cells
Authors: Liu Wenli , Schoenkerman Aaron , Lowe William L.
Published: December 2017

To better understand the molecular mechanisms for hyperglycemia-induced proatherogenic changes in endothelial cells, the effect of high glucose on activation of members of the mitogen-activated protein kinase (MAPK) family, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)-1, -2, and -5, and p38 kinase, was examined in bovine pulmonary artery endothelial cells (PAEC). Glucose, fructose, and raffinose induced a concentration-dependent decrease in PAEC growth. Addition of 25 mM glucose, fructose, or raffinose to normal growth medium stimulated an approximately twofold increase in JNK1 activity that was maximal after 24 h, whereas only glucose markedly increased ERK5 activity. Neither ERK1/2 nor p38 kinase activity was increased by glucose, fructose, or raffinose. The antioxidant N-acetylcysteine partially abrogated the glucose-induced increase in ERK5 activity but had no effect on the increase in JNK1 activity. In contrast, azaserine, which prevents increased flux through the hexosamine pathway, decreased glucose-induced JNK1 activity but had no effect on fructose- or raffinose-induced JNK1 activity. Consistent with this finding, glucosamine stimulated a 2.4-fold increase in JNK1 activity and reproduced the inhibitory effect of glucose on PAEC growth. In summary, glucose activates different members of the MAPK family in PAEC via distinct mechanisms. Moreover, the correlation between the ability of different sugars to activate JNK1 and inhibit cell growth suggests that activation of this signaling pathway may contribute to the growth inhibitory effect of glucose in endothelial cells.
Differential expression of hypothalamic neuropeptides in the early phase of diet-induced obesity in mice
Authors: Ziotopoulou Mary , Mantzoros Christos S. , Hileman Stanley M. , Flier Jeffrey S.
Published: December 2017

Exposure to high-fat diets for prolonged periods results in positive energy balance and obesity, but little is known about the initial physiological and neuroendocrine response of obesity-susceptible strains to high-fat feeding. To assess responses of C57BL/6J mice to high- and low-fat diets, we quantitated the hypothalamic expression of neuropeptides implicated in weight regulation and neuroendocrine function over a 2-wk period. Exposure to high-fat diet increased food consumption over a 2-day period during which leptin levels were increased when assessed by a frequent sampling protocol [area under the curve (AUC): 134.6 ± 10.3 vs. 100 ± 12.3, P = 0.03 during first day and 126.5 ± 8.2 vs. 100 ± 5.2, P = 0.02 during second day]. During this period, hypothalamic expression of neuropeptide Y (NPY) and agouti-related protein (AgRP) decreased by ∼30 and 50%, respectively ( P < 0.001). After 1 wk, both caloric intake and hypothalamic expression of NPY and AgRP returned toward baseline. After 2 wk, cumulative caloric intake was again higher in the high-fat group, and now proopiomelanocortin (POMC) was elevated by 76% ( P = 0.01). This study demonstrates that high-fat feeding induces hyperphagia, hyperleptinemia, and transient suppression of orexigenic neuropeptides during the first 2 days of diet. The subsequent induction of POMC may be a second defense against obesity. Attempts to understand the hypothalamic response to high-fat feeding must examine the changes as they develop over time.
Endothelial cell nitric oxide inhibits aldosterone synthesis in zona glomerulosa cells: modulation by oxygen
Authors: Hanke Craig J. , Campbell William B.
Published: December 2017

The regulation of aldosterone synthesis by endogenous nitric oxide (NO) was examined in cultured cells of the adrenal cortex. Endothelial NO synthase (eNOS) was detected by Western blot in cultured adrenal endothelial cells (ECs) but not in zona glomerulosa (ZG) cells or adrenal fibroblasts. Neither inducible (iNOS) nor neuronal NOS (nNOS) isoforms were detected in the cells. Only ECs had NOS activity and converted [3H]l-arginine to [3H]l-citrulline. Angiotensin II (ANG II, 100 nM) increased EC production of nitrate/nitrite by 2.4-fold. Coincubation with ECs or treatment with DETA nonoate increased the fluorescence of ZG cells loaded with an NO-sensitive dye, diaminofluorescein 2 diacetate (DAF-2 DA). DETA nonoate inhibited ANG II (1 nM) and potassium (10 mM) -stimulated aldosterone release in a concentration-related manner. This inhibitory effect of NO was enhanced >10-fold by decreasing the oxygen concentration from 21 to 8%. Coincubation of EC and ZG cells in 8% oxygen inhibited ANG II-induced aldosterone release, and inhibition was reversed by blockade of NOS. These findings indicate that adrenal EC-derived NO inhibits aldosterone release by cultured ZG cells and that the sensitivity to NO inhibition is increased at low oxygen concentrations.
Reduced insulin-stimulated glucose transport in denervated muscle is associated with impaired Akt-α activation
Authors: Wilkes Jason J. , Bonen Arend
Published: December 2017

Insulin signaling was examined in muscle made insulin resistant by short-term (24-h) denervation. Insulin-stimulated glucose transport in vitro was reduced by 28% ( P < 0.05) in denervated muscle (DEN). In control muscle (SHAM), insulin increased levels of surface-detectable GLUT-4 (i.e., translocated GLUT-4) 1.8-fold ( P < 0.05), whereas DEN surface GLUT-4 was not increased by insulin ( P > 0.05). Insulin treatment in vivo induced a rapid appearance of phospho[Ser473]Akt-α in SHAM 3 min after insulin injection. In DEN, phospho[Ser473]Akt-α also appeared at 3 min, but Ser473-phosphorylated Akt-α was 36% lower than in SHAM ( P < 0.05). In addition, total Akt-α protein in DEN was 37% lower than in SHAM ( P < 0.05). Akt-α kinase activity was lower in DEN at two insulin levels tested: 0.1 U insulin/rat (−22%, P < 0.05) and 1 U insulin/rat (−26%, P < 0.01). These data indicate that short-term (24-h) denervation, which lowers insulin-stimulated glucose transport, is associated with decreased Akt-α activation and impaired insulin-stimulated GLUT-4 appearance at the muscle surface.
Increased mitochondrial proton leak in skeletal muscle mitochondria of UCP1-deficient mice
Authors: Monemdjou Shadi , Hofmann Wolfgang E. , Kozak Leslie P. , Harper Mary-Ellen
Published: December 2017

Mice having targeted inactivation of uncoupling protein 1 (UCP1) are cold sensitive but not obese (Enerbäck S, Jacobsson A, Simpson EM, Guerra C, Yamashita H, Harper M-E, and Kozak LP. Nature 387: 90–94, 1997). Recently, we have shown that proton leak in brown adipose tissue (BAT) mitochondria from UCP1-deficient mice is insensitive to guanosine diphosphate (GDP), a well known inhibitor of UCP1 activity (Monemdjou S, Kozak LP, and Harper M-E. Am J Physiol Endocrinol Metab 276: E1073-E1082, 1999). Moreover, despite a fivefold increase of UCP2 mRNA in BAT of UCP1-deficient mice, we found no differences in the overall kinetics of this GDP-insensitive proton leak between UCP1 -deficient mice and controls. Based on these findings, which show no adaptive increase in UCP1-independent leak in BAT, we hypothesized that adaptive thermogenesis may be occurring in other tissues of the UCP1-deficient mouse (e.g. , skeletal muscle), thus allowing them to maintain their normal resting metabolic rate, feed efficiency, and adiposity. Here, we report on the overall kinetics of the mitochondrial proton leak, respiratory chain, and ATP turnover in skeletal muscle mitochondria from UCP1-deficient and heterozygous control mice. Over a range of mitochondrial protonmotive force (Δp) values, leak-dependent oxygen consumption is higher in UCP1-deficient mice compared with controls. State 4 (maximal leak-dependent) respiration rates are also significantly higher in the mitochondria of mice deficient in UCP1, whereas state 4 Δp is significantly lower. No significant differences in state 3 respiration rates or Δp values were detected between the two groups. Thus the altered kinetics of the mitochondrial proton leak in skeletal muscle of UCP1 -deficient mice indicate a thermogenic mechanism favoring the lean phenotype of the UCP1 -deficient mouse.
Leptin responses to glucose infusions in obesity-prone rats
Authors: Levy James R. , Lesko John , Krieg Richard J. , Adler Robert A. , Stevens Wayne
Published: December 2017

The secretion of leptin is dually regulated. In fasting animals, plasma leptin concentrations reflect body fat stores, whereas the incremental leptin response to fasting or refeeding most likely reflects insulin-mediated energy flux and metabolism within adipocytes. Impaired secretion of leptin in either pathway could result in obesity. We therefore measured plasma leptin concentrations in fasted animals and plasma leptin concentrations after an intravenous glucose infusion in a rat model of obesity. Young Sprague-Dawley (S-D) and Fischer 344 (F344) rats had similar percent body fat and fasting glucose and fasting leptin concentrations. However, F344 animals had higher insulin concentrations and leptin responses to intravenous glucose than did the S-D animals. The animals were then fed a control or high-fat diet for 6 wk. High-fat fed animals gained more weight and body fat than did the control fed animals. Control and high-fat fed F344 animals gained ∼40% ( P < 0.0001) more weight and >100% ( P < 0.01) more body fat than did the S-D animals. Fasting leptin concentrations and leptin concentrations after intravenous glucose infusions and feeding were more than double ( P < 0.05) in F344 animals compared with S-D animals. Whether an animal is fed a control or high-fat diet had little effect on the leptin response to intravenous glucose. In conclusion, young, lean F344 animals, before the onset of obesity, demonstrated a greater acute leptin response to intravenous glucose than similarly lean S-D animals. After a 6-wk diet, F344 animals had a greater percent increase in body weight and insulin resistance and exhibited higher fasting leptin concentrations and a greater absolute leptin response to intravenous glucose compared with the S-D animals. The chronic diet (control or high fat) had little impact on the acute leptin response to intravenous glucose. F344 animals exhibit leptin resistance in young, lean animals and after aging and fat accumulation.
Estimations of muscle interstitial insulin, glucose, and lactate in type 2 diabetic subjects
Authors: Sjöstrand Mikaela , Holmäng Agneta , Strindberg Lena , Lönnroth Peter
Published: December 2017

Previous measurement of insulin in human muscle has shown that interstitial muscle insulin and glucose concentrations are ∼30–50% lower than in plasma during hyperinsulinemia in normal subjects. The aims of this study were to measure interstitial muscle insulin and glucose in patients with type 2 diabetes to evaluate whether transcapillary transport is part of the peripheral insulin resistance. Ten patients with type 2 diabetes and ten healthy controls matched for sex, age, and body mass index were investigated. Plasma and interstitial insulin, glucose, and lactate (measured by intramuscular in situ-calibrated microdialysis) in the medial quadriceps femoris muscle were analyzed during a hyperinsulinemic euglycemic clamp. Blood flow in the contralateral calf was measured by vein plethysmography. At steady-state clamping, at 60–120 min, the interstitial insulin concentration was significantly lower than arterial insulin in both groups (409 ± 86 vs. 1,071 ± 99 pmol/l, P < 0.05, in controls and 584 ± 165 vs. 1,253 ± 82 pmol/l, P < 0.05, in diabetic subjects, respectively). Interstitial insulin concentrations did not differ significantly between diabetic subjects and controls. Leg blood flow was significantly higher in controls (8.1 ± 1.2 vs. 4.4 ± 0.7 ml · 100 g−1· min−1in diabetics, P < 0.05). Calculated glucose uptake was less in diabetic patients compared with controls (7.0 ± 1.2 vs. 10.8 ± 1.2 μmol · 100 g−1· min−1, P < 0.05, respectively). Arterial and interstitial lactate concentrations were both higher in the control group (1.7 ± 0.1 vs. 1.2 ± 0.1, P < 0.01, and 1.8 ± 0.1 vs. 1.2 ± 0.2 mmol/l, P < 0.05, in controls and diabetics, respectively). We conclude that, during hyperinsulinemia, muscle interstitial insulin and glucose concentrations did not differ between patients with type 2 diabetes and healthy controls despite a significantly lower leg blood flow in diabetic subjects. It is suggested that decreased glucose uptake in type 2 diabetes is caused by insulin resistance at the cellular level rather than by a deficient access of insulin and glucose surrounding the muscle cell.
Skeletal muscle apoptosis after burns is associated with activation of proapoptotic signals
Authors: Yasuhara Shingo , Perez Mary-Ellen , Kanakubo Emi , Yasuhara Yoko , Shin Yong-Sup , Kaneki Masao , Fujita Toshiro , Martyn J. A. Jeevendra
Published: December 2017

Critical illness is associated with muscle wasting and muscle weakness. Using burn injury as a model of local and systemic inflammatory response, we tested the hypothesis that thermal injury causes apoptosis in muscle. After a 40% body surface area burn to rats, abdominal muscles beneath the burn and limb muscles distant from the burn were examined for apoptosis at varying times after burn. Ladder assay, ELISA, and histological methods showed evidence of apoptosis in the abdominal muscles within 4–12 h with peak changes occurring at 3–7 days. Maximal apoptosis was also evident at distant limb muscles at 3–7 days. Investigation of proapoptotic pathways indicated mitochondrial membrane potential to be altered by 1 h after burn. Starting at 15 min after burn, cytochrome c was released from the mitochondria into the cytosol, followed by increased activity of caspase-3, starting at 6 h after burn. These studies suggest that mitochondria and caspase-mediated apoptotic pathways may be an additional mechanism of muscle weight loss in burns and may be potential therapeutic targets for prevention of muscle wasting.
IGF-I/IGFBP-3 binary complex modulates sepsis-induced inhibition of protein synthesis in skeletal muscle
Authors: Svanberg Elisabeth , Frost Robert A. , Lang Charles H. , Isgaard Jorgen , Jefferson Leonard S. , Kimball Scot R. , Vary Thomas C.
Published: December 2017

The present study evaluated the ability of insulin-like growth factor I (IGF-I) complexed with IGF binding protein-3 (IGFBP-3) to modulate the sepsis-induced inhibition of protein synthesis in gastrocnemius. Beginning 16 h after the induction of sepsis, either the binary complex or saline was injected twice daily via a tail vein, with measurements made 3 and 5 days later. By day 3, sepsis had reduced plasma IGF-I concentrations ∼50% in saline-treated rats. Administration of the binary complex provided exogenous IGF-I to compensate for the sepsis-induced diminished plasma IGF-I. Sepsis decreased rates of protein synthesis in gastrocnemius relative to controls by limiting translational efficiency. Treatment of septic rats with the binary complex for 5 days attenuated the sepsis-induced inhibition of protein synthesis and restored translational efficiency to control values. Assessment of potential mechanisms regulating translational efficiency showed that neither the sepsis-induced change in gastrocnemius content of eukaryotic initiation factor 2B (eIF2B), the amount of eIF4E associated with 4E binding protein-1 (4E-BP1), nor the phosphorylation state of 4E-BP1 or eIF4E were altered by the binary complex. Overall, the results are consistent with the hypothesis that decreases in plasma IGF-I are partially responsible for enhanced muscle catabolism during sepsis.
AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation
Authors: Chen Zhi-Ping , McConell Glenn K. , Michell Belinda J. , Snow Rodney J. , Canny Benedict J. , Kemp Bruce E.
Published: December 2017

AMP-activated protein kinase (AMPK) is a metabolic stress-sensing protein kinase responsible for coordinating metabolism and energy demand. In rodents, exercise accelerates fatty acid metabolism, enhances glucose uptake, and stimulates nitric oxide (NO) production in skeletal muscle. AMPK phosphorylates and inhibits acetyl-coenzyme A (CoA) carboxylase (ACC) and enhances GLUT-4 translocation. It has been reported that human skeletal muscle malonyl-CoA levels do not change in response to exercise, suggesting that other mechanisms besides inhibition of ACC may be operating to accelerate fatty acid oxidation. Here, we show that a 30-s bicycle sprint exercise increases the activity of the human skeletal muscle AMPK-α1 and -α2 isoforms approximately two- to threefold and the phosphorylation of ACC at Ser79(AMPK phosphorylation site) ∼8.5-fold. Under these conditions, there is also an ∼5.5-fold increase in phosphorylation of neuronal NO synthase-μ (nNOSμ) at Ser1451. These observations support the concept that inhibition of ACC is an important component in stimulating fatty acid oxidation in response to exercise and that there is coordinated regulation of nNOSμ to protect the muscle from ischemia/metabolic stress.