bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired insulin action in subcutaneous adipocytes from women with visceral obesity
Authors: Johnson Julia A. , Fried Susan K. , Pi-Sunyer F. Xavier , Albu Jeanine B.
Published: December 2017

Visceral obesity is associated with resistance to the antilipolytic effect of insulin in vivo. We investigated whether subcutaneous abdominal and gluteal adipocytes from viscerally obese women exhibit insulin resistance in vitro. Subjects were obese black and white premenopausal nondiabetic women matched for visceral adipose tissue (VAT), total adiposity, and age. Independently of race and adipocyte size, increased VAT was associated with decreased sensitivity to insulin's antilipolytic effect in subcutaneous abdominal and gluteal adipocytes. Absolute lipolytic rates at physiologically relevant concentrations of insulin or the adenosine receptor agonist N6-(phenylisopropyl)adenosine were higher in subjects with the highest vs. lowest VAT area. Independently of cell size, abdominal adipocytes were less sensitive to the antilipolytic effect of insulin than gluteal adipocytes, which may partly explain increased nonesterified fatty acid fluxes in upper vs. lower body obese women. Moreover, increased VAT was associated with decreased responsiveness, but not decreased sensitivity, to insulin's stimulatory effect on glucose transport in abdominal adipocytes. These data suggest that insulin resistance of subcutaneous abdominal and, to a lesser extent, gluteal adipocytes may contribute to increased systemic lipolysis in both black and white viscerally obese women.
Changes in uncoupling protein-2 and -3 expression in aging rat skeletal muscle, liver, and heart
Authors: Barazzoni Rocco , Nair K. Sreekumaran
Published: December 2017

Uncoupling protein (UCP)-2 and -3 mediate mitochondrial (mt) proton leak in vitro and are potential regulators of energy expenditure and ATP production. Aging is associated with alteration of tissue functions, suggesting impaired mtATP production. To determine whether age-related changes in UCP expression occur, we measured the transcript levels of UCP-2 and -3 in skeletal muscle, liver, and heart in 6- and 27-mo-old rats. UCP-2 transcripts were higher in old animals in the white (+100%) and red (+70%, both P < 0.04) gastrocnemius muscle and in the liver (+300%, P < 0.03), whereas they were comparable in the heart in both age groups. UCP-2 transcript levels correlated positively with mitochondrial-encoded cytochrome c oxidase transcripts normalized for mtDNA ( P < 0.01) and negatively with mtDNA copy number ( P < 0.001). UCP-3 transcripts were lower in the less oxidative white (−50%, P < 0.04) and unchanged in the more oxidative red (−15%, P = 0.41) gastrocnemius muscle in old animals. Similar changes at protein level were confirmed by UCP-2 protein in aging liver (+300%, P < 0.01) and UCP-2 (+85%, P < 0.05) and UCP-3 (−30%, P = 0.4) protein in aging mixed gastrocnemius muscle. Aging is thus associated with tissue-specific changes of UCP-2 and -3 gene expression. Increased UCP-2 expression may limit ATP production and is related to mitochondrial gene expression in aging muscles and liver. Different age-related changes may reflect differential regulation of UCP-2 and -3 in skeletal muscle. The current data suggest a potential role of uncoupling proteins to alter energy production in aging tissues.
Dexamethasone inhibits the stimulation of muscle protein synthesis and PHAS-I and p70 S6-kinase phosphorylation
Authors: Long Wen , Wei Liping , Barrett Eugene J.
Published: December 2017

Glucocorticoids inhibit protein synthesis in muscle. In contrast, insulin and amino acids exert anabolic actions that arise in part from their ability to phosphorylate ribosomal p70 S6-kinase (p70S6k) and eukaryotic initiation factor (eIF)4E binding protein (BP)1 (PHAS-I), proteins that regulate translation initiation. Whether glucocorticoids interfere with this action was examined by giving rats either dexamethasone (DEX, 300 μg · kg−1 · day−1, n = 10) or saline ( n = 10) for 5 days. We then measured the phosphorylation of PHAS-I and p70S6kin rectus muscle biopsies taken before and at the end of a 180-min infusion of either insulin (10 mU · min−1 · kg−1 euglycemic insulin clamp, n = 5 for both DEX- and saline-treated groups) or a balanced amino acid mixture ( n = 5 for each group also). Protein synthesis was also measured during the infusion period. The results were that DEX-treated rats had higher fasting insulin, slower glucose disposal, less lean body mass, and decreased protein synthetic rates during insulin or amino acid infusion ( P < 0.05 each). DEX did not affect basal PHAS-I or p70S6k phosphorylation but blocked insulin-stimulated phosphorylation of PHAS-I- and amino acid-stimulated phosphorylation of both PHAS-I and p70S6k ( P < 0.01, for each). DEX also increased muscle PHAS-I concentration. These effects can, in part, explain glucocorticoid-induced muscle wasting.
Flux control in the rat gastrocnemius glycogen synthesis pathway by in vivo13C/31P NMR spectroscopy
Authors: Chase J. R. , Rothman D. L. , Shulman R. G.
Published: December 2017

To determine the relative contributions of glucose transport/hexokinase, glycogen synthase (GSase), and glycolysis to the control of insulin-stimulated muscle glycogen synthesis, we combined13C and31P NMR to quantitate the glycogen synthesis rate and glucose 6-phosphate (G-6- P) levels in rat (Sprague-Dawley) gastrocnemius muscle during hyperinsulinemia at euglycemic (E) and hyperglycemic (H) glucose concentrations under thiopental anesthesia. Flux control was calculated using metabolic control analysis. The combined control coefficient of glucose transport/hexokinase (GT/Hk) for glycogen synthesis was 1.1 ± 0.03 (direct measure) and 1.14–1.16 (calculated for a range of glycolytic fluxes), whereas the control coefficient for GSase was much lower (0.011–0.448). We also observed that the increase in in vivo [G-6- P] from E to H (0.22 ± 0.03 to 0.40 ± 0.03 mM) effects a supralinear increase in the in vitro velocity of GSase, from 14.6 to 26.1 mU · kg−1· min−1(1.8-fold). All measurements suggest that the majority of the flux control of muscle glycogen synthesis is at the GT/Hk step.
Mineralocorticoid and glucocorticoid receptors inhibit UCP expression and function in brown adipocytes
Authors: Viengchareun Say , Penfornis Patrice , Zennaro Maria-Christina , Lombès Marc
Published: December 2017

Uncoupling proteins (UCP), specific mitochondrial proton transporters that function by uncoupling oxidative metabolism from ATP synthesis, are involved in thermoregulation and control of energy expenditure. The hibernoma-derived T37i cells, which possess functional endogenous mineralocorticoid receptors (MR), can undergo differentiation into brown adipocytes. In differentiated T37i cells, UCP1 mRNA levels increased 10- to 20-fold after retinoic acid or β-adrenergic treatment. Interestingly, UCP2 and UCP3 mRNA was also detected. Aldosterone treatment induced a drastic decrease in isoproterenol- and retinoic acid-stimulated UCP1 mRNA levels in a time- and dose-dependent manner (IC50≈ 1 nM aldosterone). This inhibition was unaffected by cycloheximide and did not modify UCP1 mRNA stability (half-life time = 5 h), indicating that it occurs at the transcriptional level. It involves both the MR and/or the glucocorticoid receptor (GR), depending on the retinoic or catecholamine induction pathway. Basal UCP3 expression was also significantly reduced by aldosterone, whereas UCP2 mRNA levels were not modified. Finally, as demonstrated by JC1 aggregate formation in living cells, aldosterone restored mitochondrial membrane potential abolished by isoproterenol or retinoic acid. Our results demonstrate that MR and GR inhibit expression of UCP1 and UCP3, thus participating in the control of energy expenditure.
Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE
Authors: Wautier Marie-Paule , Chappey Olivier , Corda Stefano , Stern David M. , Schmidt Ann Marie , Wautier Jean-Luc
Published: December 2017

Engagement of the receptor for advanced glycation end products (RAGE) by products of nonenzymatic glycation/oxidation triggers the generation of reactive oxygen species (ROS), thereby altering gene expression. Because dissection of the precise events by which ROS are generated via RAGE is relevant to the pathogenesis of complications in AGE-related disorders, such as diabetes and renal failure, we tested the hypothesis that activation of NADPH oxidase contributed, at least in part, to enhancing oxidant stress via RAGE. Here we show that incubation of human endothelial cells with AGEs on the surface of diabetic red blood cells, or specific AGEs, (carboxymethyl)lysine (CML)-modified adducts, prompted intracellular generation of hydrogen peroxide, cell surface expression of vascular cell adhesion molecule-1, and generation of tissue factor in a manner suppressed by treatment with diphenyliodonium, but not by inhibitors of nitric oxide. Consistent with an important role for NADPH oxidase, although macrophages derived from wild-type mice expressed enhanced levels of tissue factor upon stimulation with AGE, macrophages derived from mice deficient in a central subunit of NADPH oxidase, gp91phox, failed to display enhanced tissue factor in the presence of AGE. These findings underscore a central role of NADPH oxidase in AGE-RAGE-mediated generation of ROS and provide a mechanism for altered gene expression in AGE-related disorders.
Insulin secretion and IP levels in two distant lineages of the genusMus: comparisons with rat islets
Authors: Zawalich Walter S. , Zawalich Kathleen C. , Tesz Gregory J. , Sterpka John A. , Philbrick William M.
Published: December 2017

Islet responses of two different Mus geni, the laboratory mouse ( Mus musculus) and a phylogenetically more ancient species ( Mus caroli), were measured and compared with the responses of islets from rats ( Rattus norvegicus). A minimal and flat second-phase response to 20 mM glucose was evoked from M. musculus islets, whereas a large rising second-phase response characterized rat islets. M. caroli responses were intermediate between these two extremes; a modest rising second-phase response to 20 mM glucose was observed. Prior, brief stimulation of rat islets with 20 mM glucose results in an amplified insulin secretory response to a subsequent 20 mM glucose challenge. No such potentiation or priming was observed from M. musculus islets. In contrast, M. caroli islets displayed a modest twofold potentiated first-phase response upon subsequent restimulation with 20 mM glucose. Inositol phosphate (IP) accumulation in response to 20 mM glucose stimulation in [3H]inositol-prelabeled rat or mouse islets paralleled the insulin secretory responses. The divergence in 20 mM glucose-induced insulin release between these species may be attributable to differences in phospholipase C-mediated IP accumulation in islets.
Direct evidence for tonic sympathetic support of resting metabolic rate in healthy adult humans
Authors: Monroe Mary Beth , Seals Douglas R. , Shapiro Linda F. , Bell Christopher , Johnson David , Jones Pamela Parker
Published: December 2017

The sympathetic nervous system (SNS) plays an important role in the regulation of energy expenditure. However, whether tonic SNS activity contributes to resting metabolic rate (RMR) in healthy adult humans is controversial, with the majority of studies showing no effect. We hypothesized that an intravenous propranolol infusion designed to achieve complete β-adrenergic blockade would result in a significant acute decrease in RMR in healthy adults. RMR (ventilated hood, indirect calorimetry) was measured in 29 healthy adults (15 males, 14 females) before and during complete β-adrenergic blockade documented by plasma propranolol concentrations ≥100 ng/ml, lack of heart rate response to isoproterenol, and a plateau in RMR with increased doses of propranolol. Propranolol infusion evoked an acute decrease in RMR (−71 ± 11 kcal/day; −5 ± 0.7%, P < 0.0001), whereas RMR was unchanged from baseline levels during a saline control infusion ( P > 0.05). The response to propranolol differed from the response to saline control ( P < 0.01). The absolute and percent decreases in RMR with propranolol were modestly related to baseline plasma concentration of norepinephrine ( r = 0.38, P = 0.05; r= 0.44, P = 0.02, respectively). These findings provide direct evidence for the concept of tonic sympathetic β-adrenergic support of RMR in healthy nonobese adults.
Differential effects of insulin on peripheral and visceral tissue protein synthesis in neonatal pigs
Authors: Davis Teresa A. , Fiorotto Marta L. , Beckett Philip R. , Burrin Douglas G. , Reeds Peter J. , Wray-Cahen Diane , Nguyen Hanh V.
Published: December 2017

We recently demonstrated in neonatal pigs that, with amino acids and glucose maintained at fasting levels, the stimulation of protein synthesis in longissimus dorsi muscle with feeding can be reproduced by a physiological rise in insulin alone. In the current report, we determine whether the response of protein synthesis to insulin in the neonatal pig is 1) present in muscles of different fiber types, 2) proportional in myofibrillar and sarcoplasmic proteins, 3) associated with increased translational efficiency and ribosome number, and 4) present in other peripheral tissues and in viscera. Hyperinsulinemic-euglycemic-amino acid clamps were performed in 7- and 26-day-old pigs infused with 0, 30, 100, or 1,000 ng · kg−0.66· min−1of insulin to reproduce insulin levels present in fasted, fed, refed, and supraphysiological conditions, respectively. Tissue protein synthesis was measured using a flooding dose ofl-[4-3H]phenylalanine. Insulin increased protein synthesis in gastrocnemius muscle and, to a lesser degree, masseter muscle. The degree of stimulation of protein synthesis by insulin was similar in myofibrillar and sarcoplasmic proteins. Insulin increased translational efficiency but had no effect on ribosome number in muscle. All of these insulin-induced changes in muscle protein synthesis decreased with age. Insulin also stimulated protein synthesis in cardiac muscle and skin but not in liver, intestine, spleen, pancreas, or kidney. The results support the hypothesis that insulin mediates the feeding-induced stimulation of myofibrillar and sarcoplasmic protein synthesis in muscles of different fiber types in the neonate by increasing the efficiency of translation. However, insulin does not appear to be involved in the feeding-induced stimulation of protein synthesis in visceral tissues. Thus different mechanisms regulate the growth of peripheral and visceral tissues in the neonate.
Elevated ambient glucose induces acute inflammatory events in the microvasculature: effects of insulin
Authors: Booth Gregory , Stalker Timothy J. , Lefer Allan M. , Scalia Rosario
Published: December 2017

We employed intravital microscopy of the rat mesenteric microvasculature to study the effects of local hyperglycemia on leukocyte-endothelial cell interactions. Intraperitoneal injection of 6, 12.5, and 25 mmol/ld-glucose to the rat significantly and time-dependently increased leukocyte rolling and leukocyte adherence in, and leukocyte transmigration through mesenteric venules compared with control rats injected with Krebs-Henseleit (K-H) solution alone or given 25 mmol/ll-glucose intraperitoneally. The response elicited byd-glucose was associated with significant attenuation of endothelial nitric oxide (NO) release, as demonstrated by direct measurement of NO release in inferior vena caval segments isolated from rats exposed to 25 mmol/l d-glucose for 4 h ( P < 0.01 vs. vena caval segments from control rats). Local application of 0.05 U/min insulin for 90 min significantly attenuated glucose-induced leukocyte rolling, adherence, and migration ( P < 0.01 from 25 mmol/l d-glucose alone). Immunohistochemical localization of P-selectin expressed on endothelial surface was significantly increased 4 h after exposure of the mesenteric tissue to high ambient glucose ( P < 0.01 vs. ileal venules from rats injected with K-H solution alone or 25 mmol/l l-glucose). Insulin markedly inhibited endothelial cell surface expression of P-selectin in ileal venules exposed to elevated ambient glucose in vivo ( P < 0.01 vs. control rats injected with 25 mmol/l l-glucose). These data demonstrate that acute increases in ambient glucose comparable to those seen in diabetic patients are able to initiate an inflammatory response within the microcirculation. This inflammatory response to glucose is associated with upregulation of the endothelial cell adhesion molecule P-selectin and can be blocked by local application of insulin.
Stimulatory effects of leptin and muscle contraction on fatty acid metabolism are not additive
Authors: Lau R. , Blinn W. D. , Bonen A. , Dyck D. J.
Published: December 2017

Leptin has been shown to acutely stimulate fatty acid oxidation and triacylglycerol hydrolysis in skeletal muscle. These effects are similar to those induced by muscle contraction alone. Several studies have demonstrated that, during aerobic exercise, plasma leptin concentrations are well maintained; however, none has examined whether the stimulatory effects of leptin and contraction on muscle lipid metabolism are additive. This is the first study to examine the direct effect of leptin on lipid and carbohydrate (CHO) metabolism in isolated oxidative muscle over a range of contraction intensities. We examined the effect of leptin (10 μg/ml) on the synthesis and degradation of muscle lipid pools [phospholipid (PL), diacylglycerol (DG), triacylglycerol (TG)] and palmitate oxidation in isolated resting and contracting (2, 8, and 20 tetani/min) soleus muscles. At rest, leptin increased fatty acid oxidation (+40%, P < 0.05) and TG hydrolysis (+47%, P < 0.05), while blunting TG esterification (−20%, P < 0.05). Glucose oxidation was unaffected at rest in the presence of leptin. During tetanic contraction, fatty acid oxidation (+20–114%, P < 0.05) and TG esterification (+19–33%, P < 0.05) as well as net TG utilization (+23%, P < 0.05) were all significantly increased. However, leptin was without further effect on any of these parameters during contraction. Net utilization of intramuscular glycogen, as well as glucose oxidation, was unaffected during contraction by leptin. The findings of the present study indicate that leptin has an important influence on lipid metabolism in resting muscle, but not during contraction.
Sympathovagal imbalance in hyperthyroidism
Authors: Burggraaf J. , Tulen J. H. M. , Lalezari S. , Schoemaker R. C. , De Meyer P. H. E. M. , Meinders A. E. , Cohen A. F. , Pijl H.
Published: December 2017

We assessed sympathovagal balance in thyrotoxicosis. Fourteen patients with Graves' hyperthyroidism were studied before and after 7 days of treatment with propranolol (40 mg 3 times a day) and in the euthyroid state. Data were compared with those obtained in a group of age-, sex-, and weight-matched controls. Autonomic inputs to the heart were assessed by power spectral analysis of heart rate variability. Systemic exposure to sympathetic neurohormones was estimated on the basis of 24-h urinary catecholamine excretion. The spectral power in the high-frequency domain was considerably reduced in hyperthyroid patients, indicating diminished vagal inputs to the heart. Increased heart rate and mid-frequency/high-frequency power ratio in the presence of reduced total spectral power and increased urinary catecholamine excretion strongly suggest enhanced sympathetic inputs in thyrotoxicosis. All abnormal features of autonomic balance were completely restored to normal in the euthyroid state. β-Adrenoceptor antagonism reduced heart rate in hyperthyroid patients but did not significantly affect heart rate variability or catecholamine excretion. This is in keeping with the concept of a joint disruption of sympathetic and vagal inputs to the heart underlying changes in heart rate variability. Thus thyrotoxicosis is characterized by profound sympathovagal imbalance, brought about by increased sympathetic activity in the presence of diminished vagal tone.
Mechanism of fat-induced hepatic gluconeogenesis: effect of metformin
Authors: Song Shaoming , Andrikopoulos Sofianos , Filippis Christine , Thorburn Anne W. , Khan David , Proietto Joseph
Published: December 2017

High-fat feeding has been shown to cause hepatic insulin resistance. The aims of this study were to investigate the biochemical steps responsible for enhanced gluconeogenesis as a result of increased dietary fat intake and the site or sites at which the antihyperglycemic agent metformin acts to inhibit this process. Male Hooded Wistar rats were fed either a standard chow diet (5% fat by weight) or a high-fat diet (60% fat by weight) for 14 days with or without metformin. Total endogenous glucose production and gluconeogenesis were determined using [6-3H]glucose and [U-14C]alanine, respectively. Gluconeogenic enzyme activity and, where appropriate, protein and mRNA levels were measured in liver tissues. The high-fat diet increased endogenous glucose production (21.9 ± 4.4 vs. 32.2 ± 4.8 μmol · kg−1· min−1, P < 0.05) and alanine gluconeogenesis (4.5 ± 0.9 vs. 9.6 ± 1.9 μmol · kg−1· min−1, P < 0.05). Metformin reduced both endogenous glucose production (32.2 ± 4.8 vs. 16.1 ± 2.1 μmol · kg−1· min−1, P < 0.05) and alanine gluconeogenesis (9.6 ± 1.9 vs. 4.7 ± 0.8 μmol · kg−1· min−1, P < 0.05) after high-fat feeding. These changes were reflected in liver fructose-1,6-bisphosphatase protein levels (4.5 ± 0.9 vs. 9.6 ± 1.9 arbitrary units, P < 0.05 chow vs. high-fat feeding; 9.5 ± 1.9 vs. 4.7 ± 0.8 arbitrary units, P < 0.05 high fat fed in the absence vs. presence of metformin) but not in changes to the activity of other gluconeogenic enzymes. There was a significant positive correlation between alanine gluconeogenesis and fructose-1,6-bisphosphatase protein levels ( r = 0.56, P < 0.05). Therefore, excess supply of dietary fat stimulates alanine gluconeogenesis via an increase in fructose-1,6-bisphosphatase protein levels. Metformin predominantly inhibits alanine gluconeogenesis by preventing the fat-induced changes in fructose-1,6-bisphosphatase levels.
Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways
Authors: Carpio Luisa , Gladu Julienne , Goltzman David , Rabbani Shafaat A.
Published: December 2017

Parathyroid hormone (PTH)-related peptide (PTHrP) can modulate the proliferation and differentiation of a number of cell types including osteoblasts. PTHrP can activate a G protein-coupled PTH/PTHrP receptor, which can interface with several second-messenger systems. In the current study, we have examined the signaling pathways involved in stimulated type I collagen and alkaline phosphatase expression in the human osteoblast-derived osteosarcoma cells, MG-63. By use of Northern blotting and histochemical analysis, maximum induction of these two markers of osteoblast differentiation occurred after 8 h of treatment with 100 nM PTHrP-(1–34). Chemical inhibitors of adenylate cyclase (H-89) or of protein kinase C (chelerythrine chloride) each diminished PTHrP-mediated type I collagen and alkaline phosphatase stimulation in a dose-dependent manner. These effects of PTHrP could also be blocked by inhibiting the Ras-mitogen-activated protein kinase (MAPK) pathway with a Ras farnesylation inhibitor, B1086, or with a MAPK inhibitor, PD-98059. Transient transfection of MG-63 cells with a mutant form of Gα, which can sequester βγ-subunits, showed significant downregulation of PTHrP-stimulated type I collagen expression, as did inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) by wortmannin. Consequently, the βγ-PI 3-kinase pathway may be involved in PTHrP stimulation of Ras. Collectively, these results demonstrate that, acting via its G protein-coupled receptor, PTHrP can induce indexes of osteoblast differentiation by utilizing multiple, perhaps parallel, signaling pathways.
Fos expression in rat celiac ganglion: an index of the activation of postganglionic sympathetic nerves
Authors: Mei Qi , Mundinger Thomas O. , Kung David , Baskin Denis G. , Taborsky Gerald J.
Published: December 2017

To develop an index of the activation of abdominal sympathetic nerves, we used Fos immunostaining of the celiac ganglion (CG) taken from rats receiving nicotine, preganglionic nerve stimulation, or glucopenic agents. Subcutaneous nicotine injection moderately increased Fos expression in the principal ganglionic cells of the CG (17 ± 4 Fos+ per mm2, ∼12% of all principal CG cells), whereas subcutaneous saline had no effect (0 ± 0 Fos+ per mm2; n = 7; P < 0.01). Greater Fos expression was obtained by applying nicotine topically to the CG (71 ± 8 Fos+ per mm2; 52% of all principal CG cells, n = 5; P < 0.01 vs. topical saline, n = 4) and by preganglionic nerve stimulation (126 ± 9 Fos+ per mm2; 94% of all principal CG cells, n = 11; P < 0.01 vs. nerve isolation, n = 7). Moderate Fos expression was also observed in the CG after intraperitoneal 2-deoxy-d-glucose (2DG) injection (21 ± 2 Fos+ per mm2; 16% of all principal CG cells, n = 5; P < 0.01 vs. saline ip) or insulin injection (16 ± 2 Fos+ per mm2; 12% of all principal CG cells, n = 6; P < 0.01 vs. saline ip). Furthermore, Fos expression induced by 2DG was dose and time dependent. These data demonstrate significant Fos expression in the CG in response to chemical, electrical, and reflexive stimulation. Thus Fos expression in the CG may be a useful index to describe various levels of activation of its postganglionic sympathetic neurons.
Electrostimulation enhances FAT/CD36-mediated long-chain fatty acid uptake by isolated rat cardiac myocytes
Authors: Luiken J. J. F. P. , Willems J. , van der Vusse G. J. , Glatz J. F. C.
Published: December 2017

We investigated palmitate uptake and utilization by contracting cardiac myocytes in suspension to explore the link between long-chain fatty acid (FA) uptake and cellular metabolism, in particular the role of fatty acid translocase (FAT)/CD36-mediated transsarcolemmal FA transport. For this, an experimental setup was developed to electrically stimulate cardiomyocytes in multiple parallel incubations. Electrostimulation at voltages ≥170 V resulted in cellular contraction with no detrimental effect on cellular integrity. At 200 V and 4 Hz, palmitate uptake (measured after 3-min incubation) was enhanced 1.5-fold. In both quiescent and contracting myocytes, after their uptake, palmitate was largely and rapidly esterified, mainly into triacylglycerols. Palmitate oxidation (measured after 30 min) contributed to 22% of palmitate taken up by quiescent cardiomyocytes and, after stimulation at 4 Hz, was increased 2.8-fold to contribute to 39% of palmitate utilization. The electrostimulation-mediated increase in palmitate uptake was blocked in the presence of either verapamil, a contraction inhibitor, or sulfo- N-succinimidyl-FA esters, specific inhibitors of FAT/CD36. These data indicate that, in contracting cardiac myocytes, palmitate uptake is increased due to increased flux through FAT/CD36.
Selective tonic inhibition of G-6-Pase catalytic subunit, but not G-6-Ptransporter, gene expression by insulin in vivo
Authors: Hornbuckle Lauri A. , Edgerton Dale S. , Ayala Julio E. , Svitek Christina A. , Oeser James K. , Neal Doss W. , Cardin Sylvain , Cherrington Alan D. , O'Brien Richard M.
Published: December 2017

The regulation of glucose-6-phosphatase (G-6-Pase) catalytic subunit and glucose 6-phosphate (G-6- P) transporter gene expression by insulin in conscious dogs in vivo and in tissue culture cells in situ were compared. In pancreatic-clamped, euglycemic conscious dogs, a 5-h period of hypoinsulinemia led to a marked increase in hepatic G-6-Pase catalytic subunit mRNA; however, G-6 -P transporter mRNA was unchanged. In contrast, a 5-h period of hyperinsulinemia resulted in a suppression of both G-6-Pase catalytic subunit and G-6 -P transporter gene expression. Similarly, insulin suppressed G-6-Pase catalytic subunit and G-6 -P transporter gene expression in H4IIE hepatoma cells. However, the magnitude of the insulin effect was much greater on G-6-Pase catalytic subunit gene expression and was manifested more rapidly. Furthermore, cAMP stimulated G-6-Pase catalytic subunit expression in H4IIE cells and in primary hepatocytes but had no effect on G-6 -P transporter expression. These results suggest that the relative control strengths of the G-6-Pase catalytic subunit and G-6 -P transporter in the G-6-Pase reaction are likely to vary depending on the in vivo environment.
Analysis of recombinant Phex: an endopeptidase in search of a substrate
Authors: Guo Rong , Liu Shiguang , Spurney Robert F. , Quarles L. D.
Published: December 2017

X-linked hypophosphatemia (XLH) is caused by inactivating mutations of Phex, a phosphate-regulating endopeptidase. Further advances in our knowledge of the pathogenesis of XLH require identification of the biological function of Phex and its physiologically relevant substrates. We evaluated several potential substrates using mouse recombinant wild-type Phex proteins (r Phex-WT) and inactive mutant Phex proteins (r Phex-3′M) lacking the COOH-terminal catalytic domain as controls. By Western blot analysis, we demonstrated that Phex is a membrane-bound 100-kDa glycosylated monomer. Neither casein, a substrate for the related endopeptidase thermolysin, human stanniocalcin 1 (hSTC-1), an osteoblast-derived phosphate-regulating factor, nor FGF-23 peptide (amino acid 172–186), comprising the region mutated in autosomal dominant hypophosphatemia, was cleaved by r Phex-WT. In addition, membranes expressing r Phex-WT, r Phex-3′M, and the empty vector hydrolyzed parathyroid hormone-(1–34), indicating the lack of Phex-specific cleavage of parathyroid hormone. In contrast, r Phex-WT did display an EDTA-dependent cleavage of the neutral endopeptidase substrate [Leu]enkephalin. Further studies with wild-type and mutant r Phex proteins should permit the identification of physiologically relevant substrates involved in the pathogenesis of XLH.
Mechanisms of insulin resistance in cystic fibrosis
Authors: Hardin Dana S. , Leblanc Adrian , Marshall Gailen , Seilheimer Dan K.
Published: December 2017

Cystic fibrosis (CF) is associated with a high incidence of diabetes. Studies evaluating causes of CF-related diabetes (CFRD) have consistently documented decreased insulin secretion. In patients with CFRD, insulin sensitivity has been documented to be decreased, but controversy exists in patients with normal or impaired glucose tolerance (IGT). We undertook this study 1) to reexplore insulin sensitivity in patients with IGT and 2) to evaluate potential mechanisms of insulin resistance in CF, including GLUT-4 translocation, elevation of serum cytokines, and free fatty acid (FFA) levels. We recruited nine CF subjects with impaired glucose tolerance (IGTCF) and nine age-, gender-, and body mass index-matched control volunteers. Each underwent a hyperinsulinemic euglycemic clamp (200 mU · m−2· min−1) to measure insulin sensitivity. A muscle biopsy was obtained at maximal insulin stimulation for measure of GLUT-4 translocation with sucrose gradients. An oral glucose tolerance test and National Institutes of Health (NIH) clinical status scores were measured in all volunteers. We also measured tumor necrosis factor (TNF)-α levels and FFA in all subjects. Additionally, we report the results of TNF-α and FFA in 32 CF patients previously studied by our group. Results were that glucose disposal rate (GDR) was significantly lower in the CFIGT subjects than in controls, indicative of impaired insulin action. GLUT-4 translocation was impaired in CF and correlated with GDR. TNF-α levels were higher in all CF subjects than in controls and correlated with GDR. There was no difference in FFA between CF and control subjects. Modified NIH clinical status scores were inversely correlated with GDR and TNF-α levels. We conclude that IGTCF patients have decreased peripheral insulin sensitivity. Mechanisms include elevation of TNF-α and impaired translocation of GLUT-4.
Human adipose tissue endothelial cells promote preadipocyte proliferation
Authors: Hutley L. J. , Herington A. C. , Shurety W. , Cheung C. , Vesey D. A. , Cameron D. P. , Prins J. B.
Published: December 2017

Adipogenesis is preceded by development of a microvascular network, and optimal functioning of adipose tissue as an energy store and endocrine organ is dependent on extensive vascularization. We have examined the role of endothelial cell-derived factors that influence the proliferation of human preadipocytes. Microvascular endothelial cells and preadipocytes were isolated from human omental and subcutaneous adipose tissue biopsies by use of a developed procedure of collagenase digest, immunoselection, and differential trypsinization. Conditioned medium from microvascular endothelial cell cultures promoted the proliferation of preadipocytes ( P = <0.001) and (to a lesser extent) other cell types. No depot-specific differences in mitogenic capacity of microvascular endothelial cell medium or of preadipocyte response were observed. These results indicate that adipose tissue endothelial cells secrete soluble adipogenic factor(s).
Aging skeletal muscle mitochondria in the rat: decreased uncoupling protein-3 content
Authors: Kerner Janos , Turkaly Peter J. , Minkler Paul E. , Hoppel Charles L.
Published: December 2017

The goal of the present study was to discern the cellular mechanism(s) that contributes to the age-associated decrease in skeletal muscle aerobic capacity. Skeletal muscle mitochondrial content, a parameter of oxidative capacity, was significantly lower (25 and 20% calculated on the basis of citrate synthase and succinate dehydrogenase activities, respectively) in 24-mo-old Fischer 344 rats compared with 6-mo-old adult rats. Mitochondria isolated from skeletal muscle of both age groups had identical state 3 (ADP-stimulated) and ADP-stimulated maximal respiratory rates and phosphorylation potential (ADP-to-O ratios) with both nonlipid and lipid substrates. In contrast, mitochondria from 24-mo-old rats displayed significantly lower state 4 (ADP-limited) respiratory rates and, consequently, higher respiratory control ratios. Consistent with the tighter coupling, there was a 68% reduction in uncoupling protein-3 (UCP-3) abundance in mitochondria from elderly compared with adult rats. Congruent with the respiratory studies, there was no age-associated decrease in carnitine palmitoyltransferase I and carnitine palmitoyltransferase II activities in isolated skeletal muscle mitochondria. However, there was a small, significant decrease in tissue total carnitine content. It is concluded that the in vivo observed decrease in skeletal muscle aerobic capacity with advanced age is a consequence of the decreased mitochondrial density. On the basis of the dramatic reduction of UCP-3 content associated with decreased state 4 respiration of skeletal muscle mitochondria from elderly rats, we propose that an increased free radical production might contribute to the metabolic compromise in aging.
Developmental changes in the feeding-induced activation of the insulin-signaling pathway in neonatal pigs
Authors: Suryawan Agus , Nguyen Hanh V. , Bush Jill A. , Davis Teresa A.
Published: December 2017

In neonatal animals, feeding stimulates skeletal muscle protein synthesis, a response that declines with development. Both the magnitude of the feeding response and its developmental decline can be reproduced by insulin infusion, suggesting that an altered responsiveness to insulin is a primary determinant of the developmental decline in the stimulation of protein synthesis by feeding. In this study, 7- and 26-day-old pigs were either fasted overnight or fed porcine milk after an overnight fast. We examined the abundance and degree of tyrosine phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), and IRS-2 in skeletal muscle and, for comparison, liver. We also evaluated the association of IRS-1 and IRS-2 with phosphatidylinositol 3-kinase (PI 3-kinase). The abundance of IR protein in muscle was twofold higher at 7 than at 26 days, but IRS-1 and IRS-2 abundances were similar in muscle of 7- and 26-day-old pigs. The feeding-induced phosphorylations were greater at 7 than at 26 days of age for IR (28- vs. 13-fold), IRS-1 (14- vs. 8-fold), and IRS-2 (21- vs. 12-fold) in muscle. The associations of IRS-1 and IRS-2 with PI 3-kinase were also increased by refeeding to a greater extent at 7 than at 26 days (9- vs. 5-fold and 6- vs. 4-fold, respectively). In liver, the abundance of IR, IRS-1, and IRS-2 was similar at 7 and 26 days of age. Feeding increased the activation of IR, IRS-1, IRS-2, and PI 3-kinase in liver only twofold, and these responses were unaffected by age. Thus our findings demonstrate that the feeding-induced activation of IR, IRS-1, IRS-2, and PI 3-kinase in skeletal muscle decreases with development. Further study is needed to ascertain whether the developmental decline in the feeding-induced activation of early insulin-signaling components contributes to the developmental decline in translation initiation in skeletal muscle.
Characterization of 5′-regulatory region of human myostatin gene: regulation by dexamethasone in vitro
Authors: Ma Kun , Mallidis Con , Artaza Jorge , Taylor Wayne , Gonzalez-Cadavid Nestor , Bhasin Shalender
Published: December 2017

We cloned and characterized a 3.3-kb fragment containing the 5′-regulatory region of the human myostatin gene. The promoter sequence contains putative muscle growth response elements for glucocorticoid, androgen, thyroid hormone, myogenic differentiation factor 1, myocyte enhancer factor 2, peroxisome proliferator-activated receptor, and nuclear factor-κB. To identify sites important for myostatin's gene transcription and regulation, eight deletion constructs were placed in C2C12and L6 skeletal muscle cells. Transcriptional activity of the constructs was found to be significantly higher in myotubes compared with that of myoblasts. To investigate whether glucocorticoids regulate myostatin gene expression, we incubated both cell lines with dexamethasone. On both occasions, dexamethasone dose dependently increased both the promoter's transcriptional activity and the endogenous myostatin expression. The effects of dexamethasone were blocked when the cells were coincubated with the glucocorticoid receptor antagonist RU-486. These findings suggest that glucocorticoids upregulate myostatin expression by inducing gene transcription, possibly through a glucocorticoid receptor-mediated pathway. We speculate that glucocorticoid-associated muscle atrophy might be due in part to the upregulation of myostatin expression.
Polyunsaturated fatty acids stimulate hepatic UCP-2 expression via a PPARα-mediated pathway
Authors: Armstrong Michael B. , Towle Howard C.
Published: December 2017

The discovery of homologs of the brown fat uncoupling protein(s) (UCP) UCP-2 and UCP-3 revived the hypothesis of uncoupling protein involvement in the regulation of energy metabolism. Thus we hypothesized that UCP-2 would be regulated in the hepatocyte by fatty acids, which are known to control other energy-related metabolic processes. Treatment with 250 μM palmitic acid was without effect on UCP-2 expression, whereas 250 μM oleic acid exhibited a modest eightfold increase. Eicosapentaenoic acid (EPA), a polyunsaturated fatty acid, exerted a 50-fold upregulation of UCP-2 that was concentration dependent. This effect was seen within 12 h and was maximal by 36 h. Aspirin blocked the induction of UCP-2 by EPA, indicating involvement of the prostaglandin pathway. Hepatocytes treated with arachidonic acid, the immediate precursor to the prostaglandins, also exhibited an aspirin-inhibitable increase in UCP-2 levels, further supporting the involvement of prostaglandins in regulating hepatic UCP-2. The peroxisome proliferator-activated receptor-α (PPARα) agonist Wy-14643 stimulated UCP-2 mRNA levels as effectively as EPA. These data indicate that UCP-2 is upregulated by polyunsaturated fatty acids, potentially through a prostaglandin/PPARα-mediated pathway.
Ca2+-sensing receptor expression and PTHrP secretion in PC-3 human prostate cancer cells
Authors: Sanders Jennifer L. , Chattopadhyay Naibedya , Kifor Olga , Yamaguchi Toru , Brown Edward M.
Published: December 2017

Prostate cancer metastasizes frequently to bone. Elevated extracellular calcium concentrations ([Ca2+]o) stimulate parathyroid hormone-related protein (PTHrP) secretion from normal and malignant cells, potentially acting via the [Ca2+]o-sensing receptor (CaR). Because prostate cancers produce PTHrP, if high [Ca2+]ostimulates PTHrP secretion via the CaR, this could initiate a mechanism whereby osteolysis caused by bony metastases of prostate cancer promotes further bone resorption. We investigated whether the prostate cancer cell lines LnCaP and PC-3 express the CaR and whether polycationic CaR agonists stimulate PTHrP release. Both PC-3 and LnCaP prostate cancer cell lines expressed bona fide CaR transcripts by Northern analysis and RT-PCR and CaR protein by immunocytochemistry and Western analysis. The polycationic CaR agonists [Ca2+]o, neomycin, and spermine each concentration dependently stimulated PTHrP secretion from PC-3 cells, as measured by immunoradiometric assay, with maximal, 3.2-, 3.6-, and 4.2-fold increases, respectively. In addition, adenovirus-mediated infection of PC-3 cells with a dominant negative CaR construct attenuated high [Ca2+]o-evoked PTHrP secretion, further supporting the CaR's mediatory role in this process. Finally, pretreating PC-3 cells with transforming growth factor (TGF)-β1augmented both basal and high [Ca2+]o-stimulated PTHrP secretion. Thus, in PTHrP-secreting prostate cancers metastatic to bone, the CaR could initiate a vicious cycle, whereby PTHrP-induced bone resorption releases [Ca2+]oand TGF-β stored within bone, further increasing PTHrP release and osteolysis.
Gender differences in lipid and glucose kinetics during short-term fasting
Authors: Mittendorfer Bettina , Horowitz Jeffrey F. , Klein Samuel
Published: December 2017

Data obtained from studies conducted in animal models and humans suggest that gender affects the metabolic response to fasting. However, differences in body composition between males and females confound the interpretation of these studies, because increased adiposity itself alters the metabolic response to short-term fasting. We evaluated whole body lipid and glucose kinetics during basal (14-h fast) and short-term fasting (22-h fast) conditions in six women and six men who were matched for adiposity (24 ± 2 and 23 ± 2% body wt as fat, respectively). Substrate kinetics were measured by infusing stable isotope labeled tracers of glucose ([2H2]glucose) and glycerol ([2H5]glycerol). Basal glycerol rate of appearance (Ra) in plasma, an indicator of whole body lipolytic rate, was greater in women than in men (2.1 ± 0.2 vs. 1.5 ± 0.1 μmol · kg body wt−1· min−1; P < 0.05). However, the relative increase in glycerol Rawith continued fasting was blunted in women compared with men (40 ± 7 vs. 80 ± 4% increase; P < 0.05), resulting in similar lipolytic rates in both genders at 22 h (2.8 ± 0.2 and 2.6 ± 0.1 μmol · kg body wt−1· min−1for women and men, respectively). In contrast, glucose Rawas similar in men and women at 14 h (11 ± 0.6 vs. 12 ± 0.7 μmol · kg body wt−1· min−1) and 22 h of fasting (9 ± 0.6 vs. 10 ± 0.6 μmol · kg body wt−1· min−1). These data demonstrate the presence of sexual dimorphism in lipid, but not glucose, metabolism during both basal and short-term fasting conditions.
Troglitazone inhibits glutamine metabolism in rat mesangial cells
Authors: Routh Robert , McCarthy Kevin , Welbourne Tomas
Published: December 2017

Troglitazone is a peroxisome proliferator-activated receptor-γ agonist that has been shown to halt mesangium expansion in experimental models of type 2 diabetes mellitus and to act directly on rat mesangial cells. Because glutamine serves as the precursor for cellular biosynthetic processes, we asked whether troglitazone would inhibit mesangial cell glutamine metabolism under these conditions. Confluent monolayers of rat mesangial cells were incubated in RPMI medium in the presence of troglitazone or vehicle (DMSO). Troglitazone effected a dose-dependent reduction in glutamine utilization and in alanine formation, associated with a decrease in monolayer collagen-glycosaminoglycan content. Despite the reduced glutamine uptake, ammonium formation did not decrease, consistent with increased glutamate flux through the deamination pathway. Assayable activity of the alanine aminotransferase decreased by 63%, whereas assayable glutamate dehydrogenase remained unchanged. In control monolayers, the sum of ammonium plus alanine plus glutamate nitrogen released accounted for <75% of the glutamine nitrogen uptake. In troglitazone-treated monolayers, all of the glutamine nitrogen taken up could be accounted for as ammonium nitrogen released into the medium. These results are consonant with troglitazone reducing glutamine metabolism and specifically the transamination pathway in rat mesangial cells associated with a reduction in collagen-glycosaminoglycan content.
Mechanisms of regulation of G11α protein by dexamethasone in osteoblastic UMR 106–01 cells
Authors: Cheung Ricky , Mitchell Jane
Published: December 2017

We have previously demonstrated that glucocorticoids increased Gq/11α protein expression and phospholipase C activity in the rat osteosarcoma cell line UMR 106–01. In this study, we demonstrated that G11α is the primary Gq-subtype family member expressed in UMR cells. Dexamethasone treatment increased the expression of G11α protein in both a time- and a dose-dependent manner. Glucocorticoid treatment significantly increased the half-life of G11α protein from 20.3 to 63 h. Steady-state G11α mRNA level was also increased by glucocorticoid treatment by ∼70%. This change was not the result of changes in RNA stability but rather the result of increased transcription, because the glucocorticoid-mediated upregulation of G11α mRNA was blocked by the transcription inhibitor actinomycin D. The dexamethasone induction of G11α mRNA occurred after a time lag of 12–24 h and was blocked by the protein synthesis inhibitor cycloheximide. These results suggest that the dexamethasone-induced rise in G11α protein results primarily from changes in the degradation rate of the protein, whereas changes in G11α mRNA play a smaller role and require de novo synthesis of regulatory protein(s).
Inhibition of collagen cross-linking: effects on fibrillar collagen and ventricular diastolic function
Authors: Kato S. , Spinale F. G. , Tanaka R. , Johnson W. , Cooper G. , Zile M. R.
Published: December 2017

The fibrillar collagen network is postulated to be a primary determinant of left ventricular diastolic stiffness. This hypothesis was tested by examining the structural and physiological effects of a reduction in fibrillar collagen content and cross-linking in the intact left ventricle. Collagen cross-linking was inhibited by treating five normal adult pigs with beta-aminopropionitrile (BAPN; 10 g/day po) for 6 wk; five normal untreated pigs served as controls. Left ventricular volume, mass, and function were determined by simultaneous echocardiography and catheterization. Chamber stiffness, defined by pressure vs. volume data, and myocardial stiffness, defined by stress vs. dimension data, were determined from variably loaded beats during dextran infusion. Collagen distribution (% area) and integrity (% confluence) were determined by light microscopy. Collagen content was measured by hydroxyproline assay, and collagen cross-linking was measured by salt extraction. BAPN decreased collagen distribution (% area decreased from 12 +/- 1% in control to 7 +/- 1% in BAPN, P < 0.05), collagen integrity (% confluence decreased from 8 +/- 1% in control to 4 +/- 1% in BAPN, P < 0.05), collagen content (from 36 +/- 2 mg/g dry wt in control to 27 +/- 2 mg/g dry wt in BAPN, P < 0.05), and collagen cross-linking (extractable collagen increased from 21 +/- 2% in control to 28 +/- 2% in BAPN, P < 0.05). BAPN decreased chamber stiffness (0.13 +/- 0.02 in control to 0.06 +/- 0.01 in BAPN, P < 0.05) and myocardial stiffness (10.4 +/- 0.5 in control to 6.6 +/- 0.5 in BAPN, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)