bluuebunny/crossref_metadata_2025 huggingface dataset:
Skeletal muscle IGF-binding protein-3 and -5 expressions are age, muscle, and load dependent
Authors: Spangenburg Espen E. , Abraha Tsghe , Childs Tom E. , Pattison J. Scott , Booth Frank W.
Published: March 2015

The purpose of the current study was to examine IGFBP-3, -4, and -5 mRNA and protein expression levels as a function of muscle type, age, and regrowth from an immobilization-induced atrophy in Fischer 344 × Brown Norway rats. IGFBP-3 mRNA expression in the 4-mo-old animals was significantly higher in the red and white portions of the gastrocnemius muscle compared with the soleus muscle. However, there were no significant differences in IGFBP-3 mRNA expression among any of the muscle groups in the 30-mo-old animals. There were no significant differences in IGFBP-5 mRNA expression in any of the muscle groups, whereas in the 30-mo-old animals there was significantly less IGFBP-5 mRNA expression in the white gastrocnemius compared with the red gastrocnemius muscles. Although IGFBP-3 and -5 proteins were detected in the type I soleus muscle with Western blot analyses, no detection was observed in the type II red and white portions of the gastrocnemius muscle. Aging from adult (18 mo) to old animals (30 mo) was associated with decreases in IGFBP-3 mRNA and protein and IGFBP-5 protein only in the soleus muscle. After 10 days of recovery from 10 days of hindlimb immobilization, IGFBP-3 mRNA and protein increased in soleus muscles from young (4-mo) rats; however, only IGFBP-3 protein increased in the old (30-mo) rats. Whereas there were no changes in IGFBP-5 mRNA expression during recovery, IGFBP-5 protein in the 10-day-recovery soleus muscle did increase in the young, but not in the old, rats. Because one of the functions of IGFBPs is to modulate IGF-I action on muscle size and phenotype, it is hypothesized that IGFBP-3 and -5 proteins may have potential modulatory roles in type I fiber-dominated muscles, aging, and regrowth from atrophy.
Protein intake during hemodialysis maintains a positive whole body protein balance in chronic hemodialysis patients
Authors: Veeneman Jorden M. , Kingma Hermi A. , Boer Theo S. , Stellaard Frans , De Jong Paul E. , Reijngoud Dirk-Jan , Huisman Roel M.
Published: March 2015

Protein energy malnutrition is present in 18 to 56% of hemodialysis patients. Because hemodialysis has been regarded as a catabolic event, we studied whether consumption of a protein- and energy-enriched meal improves the whole body protein balance during dialysis in chronic hemodialysis (CHD) patients. Patients were studied on a single day between dialysis (HD− protocol) in the morning while fasting and in the afternoon while consuming six small test meals. Patients were also studied during two separate dialysis sessions (HD+ protocol). Patients were fasted during one and consumed the meals during the other. Whole body protein metabolism was studied by primed constant infusion ofl-[1-13C]valine. During HD−, feeding changed the negative whole body protein balance observed during fasting to a positive protein balance. Dialysis deepened the negative balance during fasting, whereas feeding during dialysis induced a positive balance comparable to the HD− protocol while feeding. Plasma valine concentrations during the studies were correlated with whole body protein synthesis and inversely correlated with whole body protein breakdown. We conclude that the consumption of a protein- and energy-enriched meal by CHD patients while dialyzing can strongly improve whole body protein balance, probably because of the increased amino acid concentrations in blood.
Targeting of iNOS with antisense DNA plasmid reduces cytokine-induced inhibition of osteoblastic activity
Authors: Abe Takahiro , Hikiji Hisako , Shin Wee Soo , Koshikiya Noboru , Shima Sei-ichi , Nakata Jumi , Susami Takafumi , Takato Tsuyoshi , Toyo-oka Teruhiko
Published: March 2015

Proinflammatry cytokines, tumor necrosis factor-α combined with interleukin-1β, induce excessive production of nitric oxide (NO) and its cytotoxic metabolite peroxynitrite (ONOO-) via inducible nitric oxide synthase (iNOS) in murine osteoblasts. In this study, to properly estimate the effects of antisense DNA of iNOS on osteoblastic activity, we produced transformed cell lines with antisense plasmid that specifically targets the iNOS gene for potential long-lasting inhibition. Transformed antisense cell lines were identified by 1) the detection of antisense transcripts, 2) the attenuated expression of iNOS protein, 3) the reduction of NO synthase activity, and 4) the level of NO production. These cell lines targeting iNOS, which showed decreased production of both NO and ONOO-, prevented the inhibition of osteoblastic differentiation as was assayed by the mRNA expression of type I collagen, alkaline phosphatase, osteocalcin, and Core binding factor in the presence of proinflammatory cytokines. Present results indicate that the antisense DNA plasmid of iNOS is potent to reduce the cytokine-induced inhibition of osteoblastic activity.
Intravenous angiotensin induces brain c-fosexpression and vasopressin release in the near-term ovine fetus
Authors: Shi Lijun , Hu Fang , Morrissey Paul , Yao Jiaming , Xu Zhice
Published: March 2015

The effect of intravenous angiotensin II (ANG II) on fetal brain c- fos expression and arginine vasopressin (AVP) release was studied in the near-term ovine fetus. Fetuses with chronically implanted catheters received an intravenous infusion of ANG II or saline. Fetal plasma AVP concentrations were significantly increased after the peripheral administration of ANG II, with peak levels (3-fold) at 30 min after the intravenous infusion. There was no change in fetal plasma osmolality, sodium, and hematocrit levels between the control and experimental groups or between the periods before and after the infusion of ANG II. Intravenous ANG II administration induced Fos immunoreactivity (Fos-IR) in the circumventricular organs and the median preoptic nucleus of the fetal brain. Fos-IR was also demonstrated in the fetal supraoptic nuclei (SON). Double labeling demonstrated that the AVP-containing neurons in the SON were expressing c- fos in response to intravenous ANG II. These results indicate that the peripheral ANG II in the fetus may play a significant role in stimulating the central hypothalamic-neurohypophysial system during late gestation. It supports the hypothesis that circulating ANG II may act at the fetal AVP neurons in the hypothalamus in body fluid balance via the circumventricular organs, which are situated outside the blood-brain barrier, and the central neural pathway between these two brain structures has been relatively established in utero, at least at near-term.
Effect of protein restriction on sulfur amino acid catabolism in insulin-dependent diabetes mellitus
Authors: Hamadeh Mazen J. , Hoffer L. John
Published: March 2015

Persons with conventionally treated insulin-dependent diabetes mellitus (IDDM) appear to be impaired in their ability to reduce fed-state urea production appropriately in response to dietary protein restriction (Hoffer LJ, Taveroff A, and Schiffrin A. Am J Physiol 272: E59-E67, 1997). To determine whether these conclusions apply to whole body sulfur amino acid (SAA) catabolism, we used samples from this protocol to measure daily urinary sulfate excretion and fed-state sulfate production after a high-protein test meal before and after dietary protein restriction. Eight normal subjects and six IDDM subjects treated with twice-daily intermediate- and short-acting insulin consumed a mixed test meal containing 0.50 g protein/kg after adaptation to 4 days of high protein intake (1.28 g protein/kg body wt) and again after 5 days of dietary protein restriction (0.044 g/kg). Adaptation to protein restriction decreased daily urinary sulfate and urea-N excretion by ∼80%. Over the first 24 h of protein restriction, urinary sulfate excretion decreased more than urea-N excretion for both the normal and IDDM subjects. Under conditions of a high prior protein intake, fed-state sulfate production was normal for the IDDM subjects; protein restriction reduced fed-state sulfate production by 51% (normal subjects) and 59% (IDDM subjects; not significant). We conclude that whole body SAA metabolism is normal in conventionally treated IDDM before and after dietary protein restriction. SAA catabolism, as measured by fed-state sulfate production, may be a convenient and useful method to determine the extent of whole body protein dysregulation in IDDM.
Human osteoblast-like cell proliferation induced by calcitonin-related peptides involves PKC activity
Authors: Villa I. , Dal Fiume C. , Maestroni A. , Rubinacci A. , Ravasi F. , Guidobono F.
Published: March 2015

The calcitonin peptides [calcitonin (CT), calcitonin gene-related peptide (CGRP), amylin] share many biological actions, including activity on bone cells. In the present study, CT (10−11 to 10−9 M) stimulated [3H]thymidine incorporation in primary cultures of human osteoblasts (hOB), as already demonstrated for CGRP and amylin. RT-PCR analysis showed that the calcitonin receptor and the calcitonin receptor-like receptor are both expressed in hOB. In these cells, CT (10−10 M) and amylin (10−9 M), in contrast to CGRP (10−8 M), did not increase cAMP production. All three peptides stimulated protein kinase C (PKC) activity. To evaluate PKC involvement in hOB proliferation, cells were incubated with phorbol 12,13-dibutyrate, a stimulator of PKC activity; cell proliferation was increased in a dose-dependent manner (EC50 = 3.4 × 10−8 M). Staurosporine (10−9 M), a PKC inhibitor, blocked phorbol 12,13-dibutyrate-induced PKC activity and cell proliferation. Inhibition of PKC by staurosporine also counteracted the stimulatory effect of CT, CGRP, and amylin on hOB proliferation. From these data, it is deduced that the activation of PKC is important for hOB proliferation and that it is involved in the anabolic effect of CT peptides on bone.
Fiber type-specific determinants ofVmaxfor insulin-stimulated muscle glucose uptake in vivo
Authors: Petersen Hilary Ann , Fueger Patrick T. , Bracy Deanna P. , Wasserman David H. , Halseth Amy E.
Published: March 2015

The aim of this study was to determine barriers limiting muscle glucose uptake (MGU) during increased glucose flux created by raising blood glucose in the presence of fixed insulin. The determinants of the maximal velocity ( Vmax) of MGU in muscles of different fiber types were defined. Conscious rats were studied during a 4 mU · kg−1· min−1insulin clamp with plasma glucose at 2.5, 5.5, and 8.5 mM. [U-14C]mannitol and 3- O-methyl-[3H]glucose ([3H]MG) were infused to steady-state levels ( t = −180 to 0 min). These isotope infusions were continued from 0 to 40 min with the addition of a 2-deoxy-[3H]glucose ([3H]DG) infusion. Muscles were excised at t = 40 min. Glucose metabolic index (Rg) was calculated from muscle-phosphorylated [3H]DG. [U-14C]mannitol was used to determine extracellular (EC) H2O. Glucose at the outer ([G]om) and inner ([G]im) sarcolemmal surfaces was determined by the ratio of [3H]MG in intracellular to EC H2O and muscle glucose. Rgwas comparable at the two higher glucose concentrations, suggesting that rates of uptake near Vmaxwere reached. In summary, by defining the relationship of arterial glucose to [G]omand [G]imin the presence of fixed hyperinsulinemia, it is concluded that 1) Vmaxfor MGU is limited by extracellular and intracellular barriers in type I fibers, as the sarcolemma is freely permeable to glucose; 2) Vmaxis limited in muscles with predominantly type IIb fibers by extracellular resistance and transport resistance; and 3) limits to Rgare determined by resistance at multiple steps and are better defined by distributed control rather than by a single rate-limiting step.
QUICKI is a useful index of insulin sensitivity in subjects with hypertension
Authors: Chen Hui , Sullivan Gail , Yue Lilly Q. , Katz Arie , Quon Michael J.
Published: March 2015

Insulin resistance may link disorders of metabolic homeostasis such as diabetes and obesity with disorders of hemodynamic homeostasis such as hypertension. Thus it is of interest to validate simple methods for quantifying insulin sensitivity in hypertensive patients. The quantitative insulin-sensitivity check index (QUICKI) is a novel mathematical transformation of fasting blood glucose and insulin levels. In obese and diabetic subjects, QUICKI has a significantly better linear correlation with glucose clamp determinations of insulin sensitivity than minimal-model estimates. To determine whether QUICKI is also useful in hypertensive subjects, we performed glucose clamps and frequently sampled intravenous glucose tolerance tests (FSIVGTT) on 27 hypertensive subjects taken off antihypertensive medication. Indexes of insulin sensitivity derived from glucose clamp studies (SIClamp) were compared with QUICKI, minimal-model analysis of FSIVGTTs (SIMM), and homeostasis model assessment (HOMA). The correlation between QUICKI and SIClamp ( r = 0.84) was significantly better than that between SIMM and SIClamp( r = 0.65; P < 0.028). The correlation between QUICKI and SIClamp was comparable to that between 1/HOMA and SIClamp ( r = 0.82). When studies were repeated in 14 subjects who had resumed antihypertensive medications, the percent changes in SIClamp for each of these patients were significantly correlated with percent changes in QUICKI ( r = 0.61) and HOMA ( r = −0.54) but not SIMM ( r = −0.18). We conclude that QUICKI is a simple, robust index of insulin sensitivity that is useful for evaluating and following the insulin resistance of hypertensive subjects in both research studies and clinical practice.
Potential mechanisms and consequences of cardiac triacylglycerol accumulation in insulin-resistant rats
Authors: Atkinson Laura L. , Kozak Ray , Kelly Sandra E. , Onay-Besikci Arzu , Russell James C. , Lopaschuk Gary D.
Published: March 2015

The accumulation of intracellular triacylglycerol (TG) is highly correlated with muscle insulin resistance. However, it is controversial whether the accumulation of TG is the result of increased fatty acid supply, decreased fatty acid oxidation, or both. Because abnormal fatty acid metabolism is a key contributor to the pathogenesis of diabetes-related cardiovascular dysfunction, we examined fatty acid and glucose metabolism in hearts of insulin-resistant JCR:LA-cp rats. Isolated working hearts from insulin-resistant rats had glycolytic rates that were reduced to 50% of lean control levels ( P < 0.05). Cardiac TG content was increased by 50% ( P < 0.05) in the insulin-resistant rats, but palmitate oxidation rates remained similar between the insulin-resistant and lean control rats. However, plasma fatty acids and TG levels, as well as cardiac fatty acid-binding protein (FABP) expression, were significantly increased in the insulin-resistant rats. AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac fatty acid and glucose metabolism. When activated, AMPK increases fatty acid oxidation by inhibiting acetyl-CoA carboxylase (ACC) and reducing malonyl-CoA levels, and it decreases TG content by inhibiting glycerol-3-phosphate acyltransferase (GPAT), the rate-limiting step in TG synthesis. The activation of AMPK also stimulates cardiac glucose uptake and glycolysis. We thus investigated whether a decrease in AMPK activity was responsible for the reduced cardiac glycolysis and increased TG content in the insulin-resistant rats. However, we found no significant difference in AMPK activity. We also found no significant difference in various established downstream targets of AMPK: ACC activity, malonyl-CoA levels, carnitine palmitoyltransferase I activity, or GPAT activity. We conclude that hearts from insulin-resistant JCR:LA-cp rats accumulate substantial TG as a result of increased fatty acid supply rather than from reduced fatty acid oxidation. Furthermore, the accumulation of cardiac TG is associated with a reduction in insulin-stimulated glucose metabolism.
High glucose and insulin promoteO-GlcNAc modification of proteins, including α-tubulin
Authors: Walgren Jennie L. E. , Vincent Timothy S. , Schey Kevin L. , Buse Maria G.
Published: March 2015

Increased flux through the hexosamine biosynthesis pathway has been implicated in the development of glucose-induced insulin resistance and may promote the modification of certain proteins with O-linked N-acetylglucosamine ( O-GlcNAc). L6 myotubes (a model of skeletal muscle) were incubated for 18 h in 5 or 25 mM glucose with or without 10 nM insulin. As assessed by immunoblotting with an O-GlcNAc-specific antibody, high glucose and/or insulin enhanced O-GlcNAcylation of numerous proteins, including the transcription factor Sp1, a known substrate for this modification. To identify novel proteins that may be O-GlcNAc modified in a glucose concentration/insulin-responsive manner, total cell membranes were separated by one- or two-dimensional gel electrophoresis. Selected O-GlcNAcylated proteins were identified by mass spectrometry (MS) analysis. MS sequencing of tryptic peptides identified member(s) of the heat shock protein 70 (HSP70) family and rat α-tubulin. Immunoprecipitation/immunoblot studies demonstrated several HSP70 isoforms and/or posttranslational modifications, some with selectively enhanced O-GlcNAcylation following exposure to high glucose plus insulin. In conclusion, in L6 myotubes, Sp1, membrane-associated HSP70, and α-tubulin are O-GlcNAcylated; the modification is markedly enhanced by sustained increased glucose flux.
Hypercortisolemia alters muscle protein anabolism following ingestion of essential amino acids
Authors: Paddon-Jones Douglas , Sheffield-Moore Melinda , Creson Daniel L. , Sanford Arthur P. , Wolf Steven E. , Wolfe Robert R. , Ferrando Arny A.
Published: March 2015

Debilitating injury is accompanied by hypercortisolemia, muscle wasting, and disruption of the normal anabolic response to food. We sought to determine whether acute hypercortisolemia alters muscle protein metabolism following ingestion of a potent anabolic stimulus: essential amino acids (EAA). A 27-h infusion (80 μg · kg−1 · h−1) of hydrocortisone sodium succinate mimicked cortisol (C) levels accompanying severe injury (>30 μg/dl), (C + AA; n = 6). The control group (AA) received intravenous saline ( n = 6). Femoral arteriovenous blood samples and muscle biopsies were obtained during a primed (2.0 μmol/kg) constant infusion (0.05 μmol · kg−1 · min−1) ofl-[ ring-2H5]phenylalanine before and after ingestion of 15 g of EAA. Hypercortisolemia [36.5 ± 2.1 (C + AA) vs. 9.0 ± 1.0 μg/dl (AA)] increased postabsorptive arterial, venous, and muscle intracellular phenylalanine concentrations. Hypercortisolemia also increased postabsorptive and post-EAA insulin concentrations. Net protein balance was blunted (40% lower) following EAA ingestion but remained positive for a greater period of time (60 vs. 180 min) in the C + AA group. Thus, although differences in protein metabolism were evident, EAA ingestion improved muscle protein anabolism during acute hypercortisolemia and may help minimize muscle loss following debilitating injury.
Postprandial body protein synthesis and amino acid catabolism measured with leucine and phenylalanine-tyrosine tracers
Authors: Tessari Paolo , Kiwanuka Edward , Zanetti Michela , Barazzoni Rocco
Published: March 2015

Whether phenylalanine-tyrosine (Phe-Tyr) tracers yield estimates of postprandial protein synthesis comparable to those of the widely used leucine (Leu) tracer is unclear. We measured Leu oxidation (Ox), Phe hydroxylation (Hy), and their disposal into whole body protein synthesis before and after the administration of a mixed meal (62 kJ/kg body wt, 22% of energy as protein), over 4 h in healthy subjects. Both plasma and intracellular precursor pools were used. The amino acid data were extrapolated to body protein by assuming a fixed ratio of Leu to Phe in the proteins. In the postabsorptive state, whole body protein synthesis (expressed as mg · kg−1 · min−1) was similar between Leu and Phe-Tyr tracers irrespective of the precursor pool used. After the meal, Leu Ox, Phe Hy, and body protein synthesis increased ( P ≤ 0.01 vs. basal). With the use of intracellular precursor pools, the increase of protein synthesis with Phe-Tyr (+0.51 ±0.21 mg · kg−1 · min−1) and Leu tracers (+0.57 ± 0.14) were similar ( P = not significant). In contrast, with plasma pools the increase of protein synthesis was more than twofold greater with Phe-Tyr (+1.17 ± 0.19 mg · kg−1 · min−1) than that with Leu (0.50 ± 0.13 mg · kg−1 · min−1, P < 0.01). Direct correlations were found between Leu and Ox [using both plasma and intracellular pools ( r ≤ 0.65, P ≤ 0.01)] but not between Phe and either plasma or intracellular Hy. In conclusion, 1) Phe-Tyr and Leu tracers yield comparable estimates of body protein synthesis postprandially, provided that intracellular precursor pools are used; 2) both Leu Ox and Phe Hy are stimulated by a mixed meal; 3) Phe does not correlate with Hy, which might be better related to the (unknown) portal Phe.
Visceral fat and liver fat are independent predictors of metabolic risk factors in men
Authors: Nguyen-Duy Thanh-Binh , Nichaman Milton Z. , Church Timothy S. , Blair Steven N. , Ross Robert
Published: March 2015

We examined the independent associations among abdominal adipose tissue (AT), liver fat, cardiorespiratory fitness (CRF), and lipid variables in 161 Caucasian men who had a wide variation in adiposity. We measured AT and liver fat by computed tomography and CRF by a maximal exercise test on a treadmill. Visceral AT remained a significant ( P ≤ 0.05) predictor of plasma triglycerides (TG), high-density-lipoprotein cholesterol (HDL-C), and total cholesterol (TC)/HDL-C ratio (TC/HDL-C) after statistical control for abdominal subcutaneous AT, CRF, and alcohol consumption. Abdominal subcutaneous AT was not a significant ( P ≥ 0.05) correlate of any lipid variable after control for visceral AT and CRF. Furthermore, subdivision of subcutaneous AT into deep and superficial depots did not alter these observations. Visceral AT was the strongest correlate of liver fat and remained so after control for abdominal subcutaneous AT, CRF, and alcohol consumption ( r = −0.34, P < 0.01). In contrast, abdominal subcutaneous AT and CRF were not significant ( P > 0.10) correlates of liver fat after control for visceral AT. Visceral AT remained a significant ( P < 0.01) correlate of TG, HDL-C, and TC/HDL-C independent of liver fat. However, liver fat was also a significant correlate ( P ≤ 0.05) of fasting glucose and TG independent of visceral AT. These observations reinforce the importance of visceral obesity in the pathogenesis of dyslipidemia in men, and they suggest that visceral AT and liver fat carry independent health risk.
Resistin inhibits glucose uptake in L6 cells independently of changes in insulin signaling and GLUT4 translocation
Authors: Moon Byoung , Kwan Jamie Jun-Mae , Duddy Noreen , Sweeney Gary , Begum Najma
Published: March 2015

Elevated levels of resistin have been proposed to cause insulin resistance and therefore may serve as a link between obesity and type 2 diabetes. However, its role in skeletal muscle metabolism is unknown. In this study, we examined the effect of resistin on insulin-stimulated glucose uptake and the upstream insulin-signaling components in L6 rat skeletal muscle cells that were either incubated with recombinant resistin or stably transfected with a vector containing the myc-tagged mouse resistin gene. Transfected clones expressed intracellular resistin, which was released in the medium. Incubation with recombinant resistin resulted in a dose-dependent inhibition of insulin-stimulated 2-deoxyglucose (2-DG) uptake. The inhibitory effect of resistin on insulin-stimulated 2-DG uptake was not the result of impaired GLUT4 translocation to the plasma membrane. Furthermore, resistin did not alter the insulin receptor (IR) content and its phosphorylation, nor did it affect insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation, its association with the p85 subunit of phosphatidylinositol (PI) 3-kinase, or IRS-1-associated PI 3-kinase enzymatic activity. Insulin-stimulated phosphorylation of Akt/protein kinase B-α, one of the downstream targets of PI 3-kinase and p38 MAPK phosphorylation, was also not affected by resistin. Expression of resistin also inhibited insulin-stimulated 2-DG uptake when compared with cells expressing the empty vector (L6Neo) without affecting GLUT4 translocation, GLUT1 content, and IRS-1/PI 3-kinase signaling. We conclude that resistin does not alter IR signaling but does affect insulin-stimulated glucose uptake, presumably by decreasing the intrinsic activity of cell surface glucose transporters.
A forty-year memoir of research on the regulation of glucose transport into muscle
Authors: Holloszy John O.
Published: March 2015

This historical review describes the research on the regulation of glucose transport in skeletal muscle conducted in my laboratory and in collaboration with a number of colleagues in other laboratories. This research includes studies of stimulation of glucose transport, GLUT4 translocation, and GLUT4 expression by exercise/muscle contractions, the role of Ca2+in these processes, and the interactions between the effects of exercise and insulin. Among the last are the additive effects of insulin and contractions on glucose transport and GLUT4 translocation and the increases in muscle insulin sensitivity and responsiveness induced by exercise.
Lysine synthesized by the gastrointestinal microflora of pigs is absorbed, mostly in the small intestine
Authors: Torrallardona David , Harris C. Ian , Fuller Malcolm F.
Published: March 2015

This study used a digesta transfer protocol to determine the site of absorption of lysine synthesized by the gastrointestinal microflora of pigs. Eight pigs were used, four with reentrant cannulas in the terminal ileum, two with simple T cannulas in the terminal ileum, and two intact. All pigs were given, for 5 days, the same low-protein diet that included fermentable carbohydrates. The diet of two pigs with reentrant cannulas (donor) and of the two intact (control) pigs was supplemented with15NH4Cl. The two other pigs with reentrant cannulas (acceptor pigs) and those with simple cannulas (used to supply unlabeled digesta) were given the same diet but unlabeled NH4Cl. Ileal digesta were collected continuously from all of the reentrant cannulas and kept on ice. All digesta from each donor pig were reheated and returned to the distal cannula of its companion acceptor, whose ileal digesta were discarded. Unlabeled ileal digesta from the pigs with simple cannulas were instilled into the distal cannulas of the donor pigs. At the end of the experiment, the average15N enrichment in the plasma free lysine of control pigs was 0.0407 atom % excess (APE); that of donor pigs was 0.0322 APE (79% of controls), whereas that of acceptor pigs was only 0.0096 APE (24% of controls). Due to nitrogen recycling, acceptor pigs had labeled lysine in the digesta of the stomach and small intestine, and donor pigs had labeled lysine in the digesta of the large intestine. If account is taken of the higher 15N enrichment of microbial lysine in the large compared with the small intestine, it can be estimated that >90% of the absorption of microbial lysine took place in the small intestine.
Validation and calibration of DEXA body composition in mice
Authors: Brommage Robert
Published: March 2015

Validated methods of determining murine body composition are required for studies of obesity in mice. Dual-energy X-ray absorptiometry (DEXA) provides a noninvasive approach to assess body fat and lean tissue contents. Similar to DEXA analyses in other species, body fat measurements in mice show acceptable precision but suffer from poor accuracy. Because fat and lean tissues each contain various components, these inaccuracies likely result from selection of inappropriate calibration standards. Analysis of solvents showed that the PIXImus2 DEXA gave results consistent with theoretical calculations. Male mice weighing 26-60 g and having body fat percentages ranging from 3 to 49% were analyzed by both PIXImus2 DEXA and chemical carcass analysis. DEXA overestimated mouse fat content by an average of 3.3 g, and algorithms were generated to calculate body fat from both measured body fat values and the measured ratio of high- to low-energy X-ray attenuations. With calibration to mouse body fat content measured by carcass analysis, the PIXImus2 DEXA gives accurate body composition values in mice.
Effect of tumor removal on mucosal protein synthesis in patients with colorectal cancer
Authors: Rittler Peter , Demmelmair Hans , Koletzko Berthold , Jauch Karl-Walter , Hartl Wolfgang H.
Published: March 2015

It is currently controversial whether mucosal hyperproliferation is involved in colorectal cancerogenesis. The purpose of the present study was to examine protein synthetic rate as an indicator of potential tissue proliferation in grossly normal rectal mucosa from cancer-bearing subjects and to compare this rate with that in mucosa from subjects posttumor removal. Six postabsorptive patients with localized rectal cancer and five postsurgical control subjects received a primed constant infusion of [1-13C]leucine (0.16 μmol/kg min, 9.6 μmol/kg prime). Forceps biopsies from the mucosa were taken after 3 and 6 h. Protein synthesis was calculated from protein-bound leucine enrichment (determined by capillary GC-combustion IRMS) and from the enrichment of free intracellular leucine (determined by GC-quadrupole MS). In cancer-bearing subjects, mucosal protein synthesis amounted to 1.28 ± 0.24%/h. This rate was significantly higher ( P < 0.05) than the corresponding rate of mucosa from patients after cancer removal (0.69 ± 0.09%/h). These findings do not support the concept that colorectal cancer originates from a proliferative disease of the whole colon. Increased mucosal protein synthesis appears to depend on the presence of the tumor itself and should therefore be considered a secondary phenomenon.
Measuring gluconeogenesis using a low dose of2H2O: advantage of isotope fractionation during gas chromatography
Authors: Katanik Jill , McCabe Brendan J. , Brunengraber Daniel Z. , Chandramouli Visvanathan , Nishiyama Fumie J. , Anderson Vernon E. , Previs Stephen F.
Published: March 2015

The contribution of gluconeogenesis to glucose production can be measured by enriching body water with2H2O to ∼0.5% 2H and determining the ratio of 2H that is bound to carbon-5 vs. carbon-2 of blood glucose. This labeling ratio can be measured using gas chromatography-mass spectrometry after the corresponding glucose carbons are converted to formaldehyde and then to hexamethylenetetramine (HMT). We present a technique for integrating ion chromatograms that allows one to use only 0.05% 2H in body water (i.e., 10 times less than the current dose). This technique takes advantage of the difference in gas chromatographic retention times of naturally labeled HMT and [2H]HMT. We discuss the advantage(s) of using a low dose of 2H2O to quantify the contribution of gluconeogenesis.
Intravenous estrogens increase insulin clearance and action in postmenopausal women
Authors: Van Pelt R. E. , Gozansky W. S. , Schwartz R. S. , Kohrt W. M.
Published: March 2015

To test the hypothesis that estrogens alter insulin action, we evaluated the effects of intravenous conjugated estrogens (CE) on insulin-stimulated steady-state glucose infusion rate (SSGIR) and suppression of plasma glycerol in postmenopausal women (mean ± SD; 56 ± 4 yr; n = 12) not using hormone replacement. SSGIR and glycerol were measured during a two-stage (8 and 40 mU · m-2 · min-1) hyperinsulinemic euglycemic clamp on 2 days, with or without a 2.5-mg intravenous CE bolus. Serum estradiol concentrations were increased ∼200% on the estrogen (EST) compared with the control (CON) days. Serum insulin was reduced ( P < 0.01) during stage 2 of the clamp for EST (63.3 ± 12.8 μU/ml) vs. CON (78.2 ± 15.8 μU/ml). Mean SSGIR and plasma glycerol did not differ between CON and EST days. With adjustment for differences in insulin concentration between conditions, stage 2 glucose disposals were significantly higher (8.63 vs. 7.20 mg · kg-1 · min-1) and plasma glycerol concentrations were significantly lower (29.4 vs. 35.0 μmol/l) for EST vs. CON. Our findings suggest that acute CE administration increases insulin clearance and action in postmenopausal women.
Characterization of insulin signaling in rat retina in vivo and ex vivo
Authors: Reiter Chad E. N. , Sandirasegarane Lakshman , Wolpert Ellen B. , Klinger Marianne , Simpson Ian A. , Barber Alistair J. , Antonetti David A. , Kester Mark , Gardner Thomas W.
Published: March 2015

Insulin receptor (IR) signaling cascades have been studied in many tissues, but retinal insulin action has received little attention. Retinal IR signaling and activity were investigated in vivo in rats that were freely fed, fasted, or injected with insulin by phosphotyrosine immunoblotting and by measuring kinase activity. A retina explant system was utilized to investigate the IR signaling cascade, and immunohistochemistry was used to determine which retinal cell layers respond to insulin. Basal IR activity in the retina was equivalent to that in brain and significantly greater than that of liver, and it remained constant between freely fed and fasted rats. Furthermore, IR signaling increased in the retina after portal vein administration of supraphysiological doses of insulin. Ex vivo retinas responded to 10 nM insulin with IR β-subunit (IRβ) and IR substrate-2 (IRS-2) tyrosine phosphorylation and AktSer473phosphorylation. The retina expresses mRNA for all three Akt isoforms as determined by in situ hybridization, and insulin specifically increases Akt-1 kinase activity. Phospho-AktSer473immunoreactivity increases in retinal nuclear cell layers with insulin treatment. These results demonstrate that the retinal IR signaling cascade to Akt-1 possesses constitutive activity, and that exogenous insulin further stimulates this prosurvival pathway. These findings may have implications in understanding normal and dysfunctional retinal physiology.
Recent advances in mechanisms regulating glucose oxidation at the level of the pyruvate dehydrogenase complex by PDKs
Authors: Sugden Mary C. , Holness Mark J.
Published: March 2015

The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate, linking glycolysis to the tricarboxylic acid cycle and fatty acid (FA) synthesis. Knowledge of the mechanisms that regulate PDC activity is important, because PDC inactivation is crucial for glucose conservation when glucose is scarce, whereas adequate PDC activity is required to allow both ATP and FA production from glucose. The mechanisms that control mammalian PDC activity include its phosphorylation (inactivation) by a family of pyruvate dehydrogenase kinases (PDKs 1–4) and its dephosphorylation (activation, reactivation) by the pyruvate dehydrogenase phosphate phosphatases (PDPs 1 and 2). Isoform-specific differences in kinetic parameters, regulation, and phosphorylation site specificity of the PDKs introduce variations in the regulation of PDC activity in differing endocrine and metabolic states. In this review, we summarize recent significant advances in our knowledge of the mechanisms regulating PDC with emphasis on the PDKs, in particular PDK4, whose expression is linked with sustained changes in tissue lipid handling and which may represent an attractive target for pharmacological interventions aimed at modulating whole body glucose, lipid, and lactate homeostasis in disease states.
Regulation of translation initiation by insulin and amino acids in skeletal muscle of neonatal pigs
Authors: O'Connor Pamela M. J. , Kimball Scot R. , Suryawan Agus , Bush Jill A. , Nguyen Hanh V. , Jefferson Leonard S. , Davis Teresa A.
Published: March 2015

Previous studies have shown that intravenous infusion of insulin and/or amino acids reproduces the feeding-induced stimulation of muscle protein synthesis in neonates and that insulin and amino acids act independently to produce this effect. The goal of the present study was to delineate the regulatory roles of insulin and amino acids on muscle protein synthesis in neonates by examining translational control mechanisms, specifically the eukaryotic translation initiation factors (eIFs), which enable coupling of initiator methionyl-tRNAiand mRNA to the 40S ribosomal subunit. Insulin secretion was blocked by somatostatin in fasted 7-day-old pigs ( n = 8–12/group), insulin was infused to achieve plasma levels of ∼0, 2, 6, and 30 μU/ml, and amino acids were clamped at fasting or fed levels or, at the high insulin dose, below fasting. Both insulin and amino acids increased the phosphorylation of ribosomal protein S6 kinase (S6K1) and the eIF4E-binding protein (4E-BP1), decreased the binding of 4E-BP1 to eIF4E, increased eIF4E binding to eIF4G, and increased fractional protein synthesis rates but did not affect eIF2B activity. In the absence of insulin, amino acids had no effect on these translation initiation factors but increased the protein synthesis rates. Raising insulin from below fasting to fasting levels generally did not alter translation initiation factor activity but raised protein synthesis rates. The phosphorylation of S6K1 and 4E-BP1 and the amount of 4E-BP1 bound to eIF4E and eIF4E bound to eIF4G were correlated with insulin level, amino acid level, and protein synthesis rate. Thus insulin and amino acids regulate muscle protein synthesis in skeletal muscle of neonates by modulating the availability of eIF4E for 48S ribosomal complex assembly, although other processes also must be involved.
Altered cardiovascular regulation in arginine vasopressin-overexpressing transgenic rat
Authors: Tachikawa Kazushige , Yokoi Hisashi , Nagasaki Hiroshi , Arima Hiroshi , Murase Takashi , Sugimura Yoshihisa , Miura Yoshitaka , Hirabayashi Masumi , Oiso Yutaka
Published: March 2015

Although arginine vasopressin (AVP), an antidiuretic hormone, has been widely acknowledged to play an important role in cardiovascular regulation via V1a receptors (V1aR), its precise significance remains unclear. In this study, we investigated the effects of long-standing high plasma AVP status on cardiovascular regulation in the AVP-overexpressing transgenic (Tg) rat. Adult male homozygous Tg rats were compared with age-matched normal Sprague-Dawley rats as controls. There were no significant differences in mean arterial blood pressure (BP; MABP) or heart rate between Tg and control rats in the basal state. Subcutaneous injection of AVP significantly increased MABP in controls but did not cause any apparent increase in MABP in Tg rats. BP recovery from hemorrhage-induced hypotension was significantly delayed in Tg compared with control rats. Pretreatment with a selective V1aR antagonist, OPC-21268, which is thought to restore the downregulation of V1aR, markedly improved both of these impaired responses. Northern blot analysis confirmed that decreased expression of V1aR mRNA and pretreatment with V1aR antagonist significantly restored the downregulation of V1aR mRNA. These results suggest that the Tg rat has decreased sensitivity to the hypertensive effect of AVP due to downregulation of V1aR, which may function as an adaptive mechanism to maintain normal BP against chronic hypervasopressinemia. In addition, impaired restoration of BP after hemorrhage-induced hypotension in Tg rats supports a physiological role of AVP in cardiovascular regulation.
Regulation of type II deiodinase expression by EGF and glucocorticoid in HC11 mouse mammary epithelium
Authors: Song Shigeaki , Oka Takami
Published: March 2015

Thyroid hormones are important for mammary gland growth and development. The iodothyronine deiodinases play a key role in thyroid hormone metabolism. We have showed that type II 5′-deiodinase (5′D2) activity and mRNA are present in the mouse mammary gland and that their levels are reduced in the lactating gland. To investigate the regulatory mechanism of mouse 5′D2 gene ( mdio2) expression in mammary epithelium, we employed the HC11 cell line, which is derived from mouse mammary epithelial cells and retains the ability to express differentiated function. HC11 cells were treated with combinations of insulin, glucocorticoid (GC, dexamethasone), prolactin, and epidermal growth factor (EGF), and 5′D2 activity and the D2-to-GAPDH mRNA ratio were measured by125I−release from125I-labeled thyroxine and semiquantitative RT-PCR, respectively. EGF increased both 5′D2 activity and mRNA levels about twofold. GC reduced both 5′D2 activity and mRNA in a dose-dependent manner, and their levels were decreased to approximately one-tenth and one-fifth, respectively, of control levels. These data demonstrated that mdio2expression in HC11 cells is upregulated by EGF mainly at the pretranslational level and downregulated by GC at both pre- and posttranslational levels. Furthermore, we showed that GC reduced the promoter activity of the 627- bp 5′-upstream region of the mdio2/luciferase chimeric reporter gene, suggesting that GC exerts its effect, at least in part, at the transcriptional level.
Gastrin enhances gastric mucosal integrity through cyclooxygenase-2 upregulation in rats
Authors: Komori Masato , Tsuji Shingo , Sun Wei-Hao , Tsujii Masahiko , Kawai Naoki , Yasumaru Masakazu , Kakiuchi Yoshimi , Kimura Arata , Sasaki Yutaka , Higashiyama Shigeki , Kawano Sunao , Hori Masatsugu
Published: March 2015

Gastrin, PGs, and growth factors have important roles in maintaining gastrointestinal mucosal integrity. Cyclooxygenases (COX-1 and COX-2) are the key enzymes involved in PG synthesis. This study aimed to clarify the mechanisms of gastric mucosal protection by gastrin. Fasted rats were administered subcutaneous gastrin 17 with or without gastrin receptor antagonist YM022 pretreatment. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) and COX-2 expression were examined using Western blot analysis. Another series of experiments investigated 1) PGE2 levels in gastric mucosa, 2) the protective action of gastrin against gastric damage by acidified ethanol, 3) the effects of a specific HB-EGF-neutralizing antibody on gastrin-induced COX-2 expression, and 4) the effects of a specific COX-2 inhibitor NS-398 on PGE2 synthesis and the mucosal protection afforded by gastrin. Gastrin dose-dependently increased HB-EGF, COX-2 expression, and PGE2 levels and reduced gastric damage. However, pretreatment with YM022 dose-dependently abolished such effects of gastrin. A specific HB-EGF- neutralizing antibody and an EGF receptor inhibitor decreased gastrin-induced COX-2 expression. NS-398 blocked gastrin-induced PGE2 synthesis and mucosal protection. In conclusion, this study demonstrates that gastrin enhances gastric mucosal integrity through COX-2, which is partially mediated by HB-EGF, and PGE2 upregulation in rats.
Role ofpp60c-srcandp44/42MAPK in ANG II-induced contraction of rat tonic gastrointestinal smooth muscles
Authors: Puri Rajinder N. , Fan Ya-Ping , Rattan Satish
Published: March 2015

We examined the role of mitogen-activated protein kinase ( p44/42MAPK) in ANG II-induced contraction of lower esophageal sphincter (LES) and internal anal sphincter (IAS) smooth muscles. Studies were performed in the isolated smooth muscles and cells (SMC). ANG II-induced changes in the levels of phosphorylation of different signal transduction and effector proteins were determined before and after selective inhibitors. ANG II-induced contraction of the rat LES and IAS SMC was inhibited by genistein, PD-98059 [a specific inhibitor of MAPK kinases (MEK 1/2)], herbimycin A (a pp60c-srcinhibitor), and antibodies to pp60c-srcand p120ras GTPase-activating protein ( p120rasGAP). ANG II-induced contraction of the tonic smooth muscles was accompanied by an increase in tyrosine phosphorylation of p120rasGAP. These were attenuated by genistein but not by PD-98059. ANG II-induced increase in phosphorylations of p44/42MAPKs and caldesmon was attenuated by both genistein and PD-98059. We conclude that pp60c-srcand p44/42MAPKs play an important role in ANG II-induced contraction of LES and IAS smooth muscles.
Basolateral sorting signal of the 300-kDa mannose 6-phosphate receptor
Authors: Wick Debra A. , Seetharam Bellur , Dahms Nancy M.
Published: March 2015

In polarized cells, the delivery of numerous membrane proteins from the trans-Golgi network to the basolateral surface depends on specific sequences located in their cytoplasmic domain. We have previously shown that the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) exhibits a polarized cell surface distribution in the human colon adenocarcinoma (Caco-2) cell line in which there is a threefold enrichment on the basolateral surface. To investigate the role of residues in the cytoplasmic region of the receptor that facilitates its entry into the basolateral sorting pathway, we generated stably transfected Caco-2 cell lines expressing various mutant bovine IGF-II/MPRs. The steady-state surface distribution of mutant receptors was analyzed by subjecting filter-grown cell monolayers to incubation with iodinated IGF-II/MPR-specific antibody or to indirect immunofluorescence and visualization by confocal microscopy. Together, these results demonstrate that the sorting of the IGF-II/MPR to the basolateral cell surface depends on recognition of sequences located in its cytoplasmic region that are distinct from the Tyr-based internalization and dileucine-dependent endosomal trafficking motifs.
Analysis of human cellular retinol-binding protein II promoter during enterocyte differentiation
Authors: Zhang Liang , E Xueping , Luker Kathryn E. , Shao Jian-Su , Levin Marc S. , Suh Eunran , Li Ellen
Published: March 2015

Cellular retinol binding protein II (CRBP II) is a vitamin A-binding protein that is expressed specifically in small intestinal villus absorptive cells. Previous studies have shown that retinoic acid upregulates endogenous human CRBP II gene expression in differentiated Caco-2 cells. To better characterize the regulation of human CRBP II expression, we analyzed the ability of receptor-selective agonists to enhance transcription from the 5′-upstream flanking region of the human CRBP II gene. Stable transfection experiments showed that the proximal 2.8-kb region of the human CRBP II gene is sufficient for retinoic acid inducibility in differentiated Caco-2 cells. However, direct sequence analysis and transient transfection experiments indicate that, unlike the rat CRBP II promoter, the human CRBP II promoter is not a direct retinoid X receptor target. The results indicate that the retinoic acid responsiveness of the human CRBP II promoter is mediated by an indirect mechanism and that this mechanism is associated with enterocyte differentiation.
I. Nuclear receptors and bile acid homeostasis
Authors: Goodwin Bryan , Kliewer Steven A.
Published: March 2015

Bile acids are required for the absorption of lipids and fat-soluble vitamins. The hepatic biosynthesis of bile acids is a major pathway for the catabolism and removal of cholesterol from the body. Because of their intrinsic toxicity, bile acid synthesis, transport, and metabolism must be tightly regulated. It is now apparent that members of the nuclear receptor family of lipid-activated transcription factors are key regulators of these physiological processes. A greater understanding of these receptors should afford novel opportunities for therapeutic intervention in chronic diseases such as cholestasis and dyslipidemia.