bluuebunny/crossref_metadata_2025 huggingface dataset:
Unlike insulin, amino acids stimulate p70S6Kbut not GSK-3 or glycogen synthase in human skeletal muscle
Authors: Liu Zhenqi , Wu Yangsong , Nicklas Edward W. , Jahn Linda A. , Price Wendie J. , Barrett Eugene J.
Published: March 2004

Insulin stimulates muscle glucose disposal via both glycolysis and glycogen synthesis. Insulin activates glycogen synthase (GS) in skeletal muscle by phosphorylating PKB (or Akt), which in turn phosphorylates and inactivates glycogen synthase kinase 3 (GSK-3), with subsequent activation of GS. A rapamycin-sensitive pathway, most likely acting via ribosomal 70-kDa protein S6 kinase (p70S6K), has also been implicated in the regulation of GSK-3 and GS by insulin. Amino acids potently stimulate p70S6K, and recent studies on cultured muscle cells suggest that amino acids also inactivate GSK-3 and/or activate GS via activating p70S6K. To assess the physiological relevance of these findings to normal human physiology, we compared the effects of amino acids and insulin on whole body glucose disposal, p70S6K, and GSK-3 phosphorylation, and on the activity of GS in vivo in skeletal muscle of 24 healthy human volunteers. After an overnight fast, subjects received intravenously either a mixed amino acid solution (1.26 μmol·kg-1·min-1× 6 h, n = 9), a physiological dose of insulin (1 mU·kg-1·min-1euglycemic hyperinsulinemic clamp × 2 h, n = 6), or a pharmacological dose of insulin (20 mU·kg-1·min-1euglycemic hyperinsulinemic clamp × 2 h, n = 9). Whole body glucose disposal rates were assessed by calculating the steady-state glucose infusion rates, and vastus lateralis muscle was biopsied before and at the end of the infusion. Both amino acid infusion and physiological hyperinsulinemia enhanced p70S6Kphosphorylation without affecting GSK-3 phosphorylation, but only physiological hyperinsulinemia also increased whole body glucose disposal and GS activity. In contrast, a pharmacological dose of insulin significantly increased whole body glucose disposal, p70S6K, GSK-3 phosphorylation, and GS activity. We conclude that amino acids at physiological concentrations mediate p70S6Kbut, unlike insulin, do not regulate GSK-3 and GS phosphorylation/activity in human skeletal muscle.
Effects of hyperglycemia on hepatic gluconeogenic flux during glycogen phosphorylase inhibition in the conscious dog
Authors: Edgerton Dale S. , Cardin Sylvain , Neal Doss , Farmer Ben , Lautz Margaret , Pan Catherine , Cherrington Alan D.
Published: March 2004

The aim of these studies was to investigate the effect of hyperglycemia with or without hyperinsulinemia on hepatic gluconeogenic flux, with the hypothesis that inhibition would be greatest with combined hyperglycemia/hyperinsulinemia. A glycogen phosphorylase inhibitor (BAY R3401) was used to inhibit glycogen breakdown in the conscious overnight-fasted dog, and the effects of a twofold rise in plasma glucose level (HI group) accompanied by 1) euinsulinemia (HG group) or 2) a fourfold rise in plasma insulin were assessed over a 5-h experimental period. Hormone levels were controlled using somatostatin with portal insulin and glucagon infusion. In the HG group, net hepatic glucose uptake and net hepatic lactate output substantially increased. There was little or no effect on the net hepatic uptake of gluconeogenic precursors other than lactate (amino acids and glycerol) or on the net hepatic uptake of free fatty acids compared with the control group. Consequently, whereas hyperglycemia had little effect on gluconeogenic flux to glucose 6-phosphate (G-6- P), net hepatic gluconeogenic flux was reduced because of increased hepatic glycolytic flux during hyperglycemia. Net hepatic glycogen synthesis was increased by hyperglycemia. The effect of hyperglycemia on gluconeogenic flux to G-6- P and net hepatic gluconeogenic flux was similar. We conclude that, in the absence of appreciable glycogen breakdown, the increase in glycolytic flux that accompanies hyperglycemia results in decreased net carbon flux to G-6- P but no effect on gluconeogenic flux to G-6- P.
Integration of hormone signaling in the regulation of human 25(OH)D324-hydroxylase transcription
Authors: Barletta Frank , Dhawan Puneet , Christakos Sylvia
Published: March 2004

The current study sought to define the molecular mechanisms involved in the cross talk between 1,25-dihydroxyvitamin D3[1,25(OH)2D3] and activators of PKC in the regulation of 25(OH)D324-hydroxlyase [24(OH)ase]. Transfection of the h24(OH)ase promoter construct [-5,500/-22 luciferase; vitamin D response elements at -294/-274 and -174/-151; AP-1 site at -1,167/-1,160] in vitamin D receptor (VDR)-transfected COS-7 cells resulted in strong activation by 1,25(OH)2D3. In these cells, cotreatment with the PKC activator TPA and 1,25(OH)2D3yielded a 27-fold increase in luciferase activity, which was 2- to 3-fold greater than activation obtained with 1,25(OH)2D3alone ( P < 0.05). Similar results were observed using LLCPK-1 kidney cells, suggesting that the previously observed enhancement of 1,25(OH)2D3-induced renal 24(OH)ase mRNA and activity by PKC activation occurs at the level of transcription. The functional cooperation between PKC activation and VDR was not found to be mediated by the AP-1 site in the h24(OH)ase promoter or by enhanced binding of GRIP or DRIP205 to VDR and was also not due to PKC-mediated phosphorylation of VDR on Ser51. Our study demonstrates that, in LLCPK-1 kidney cells, the PKC enhancement of 1,25(OH)2D3-stimulated transcription may be due, in part, to an increase in VDR concentration. In addition, inhibitors of the MAPK pathway were found to decrease the TPA enhancement ( P < 0.05). Because activation of MAPK has been reported to result in the phosphorylation of SRC-1 and in functional cooperation between SRC-1 and CREB binding protein, we propose that the potentiation of VDR-mediated transcription may also be mediated through changes in the phosphorylation of specific VDR coregulators.
Protein calorie restriction has opposite effects on glucose metabolism and insulin gene expression in fetal and adult rat endocrine pancreas
Authors: Martín M. A. , Fernández E. , Pascual-Leone A. M. , Escrivá F. , Alvarez C.
Published: March 2004

We previously demonstrated that fetuses from undernourished pregnant rats exhibited increased β-cell mass and hyperinsulinemia, whereas keeping food restriction until adult age caused reduced β-cell mass, hypoinsulinemia, and decreased insulin secretion. Because these alterations can be related to insulin availability, we have now investigated early and long-term effects of protein calorie food restriction on insulin mRNA levels as well as the possible mechanisms that could modulate the endogenous insulin mRNA content. We used fetuses at 21.5 days of gestation proceeding from food-restricted rats during the last week of pregnancy and 70-day-old rats undernourished from day 14 of gestation until adult age and with respective controls. Insulin mRNA levels, glucose transporters, and total glycolysis and mitochondrial oxidative fluxes were evaluated. We additionally analyzed undernutrition effects on signals implicated in glucose-mediated insulin gene expression, especially pancreatic duodenal homeobox-1 (PDX-1), stress-activated protein kinase-2 (p38/SAPK2), and phosphatidylinositol 3-kinase. Undernourished fetuses showed increased insulin mRNA, oxidative glucose metabolism, and p38/SAPK2 levels, whereas undernutrition until adult age provoked a decrease in insulin gene expression, oxidative glucose metabolism, and PDX-1 levels. The results indicate that food restriction caused changes in insulin gene expression and content leading to alterations in glucose-stimulated insulin secretion. The molecular events, increased p38/SAPK2 levels in fetuses and decreased PDX-1 levels in adults, seem to be the responsible for the altered insulin mRNA expression. Moreover, because PDX-1 activation appears to be regulated by glucose-derived metabolite(s), the altered glucose oxidation caused by undernutrition could in some manner affect insulin mRNA expression.
Extremity hyperinsulinemia stimulates muscle protein synthesis in severely injured patients
Authors: Gore Dennis C. , Wolf Steven E. , Sanford Arthur P. , Herndon David N. , Wolfe Robert R.
Published: March 2004

Insulin has a well-recognized anabolic effect on muscle protein, yet critically ill, severely injured patients are often considered “resistant” to the action of insulin. The purpose of this study was to assess the in vivo effects of hyperinsulinemia on human skeletal muscle in severely injured patients. To accomplish this goal, 14 patients with burns encompassing >40% of their body surface area underwent metabolic evaluation utilizing isotopic dilution of phenylalanine, femoral artery and vein blood sampling, and sequential muscle biopsies of the leg. After baseline metabolic measurements were taken, insulin was infused into the femoral artery at 0.45 mIU·min-1·100 ml leg volume-1 to create a local hyperinsulinemia but with minimal systemic perturbations. Insulin administration increased femoral venous concentration of insulin ( P < 0.01) but with only a 4% (insignificant) decrease in the arterial glucose concentration and a 7% (insignificant) decrease in the arterial concentration of phenylalanine. Extremity hyperinsulinemia significantly increased leg blood flow ( P < 0.05) and the rate of muscle protein synthesis ( P < 0.05). Neither the rate of muscle protein breakdown nor the rate of transmembrane transport of phenylalanine was significantly altered with extremity hyperinsulinemia. In conclusion, this study demonstrates that insulin directly stimulates muscle protein synthesis in severely injured patients.
Quantifying rates of protein synthesis in humans by use of2H2O: application to patients with end-stage renal disease
Authors: Previs Stephen F. , Fatica Richard , Chandramouli Visvanathan , Alexander James C. , Brunengraber Henri , Landau Bernard R.
Published: March 2004

A method is introduced for quantitating protein synthetic rates in humans by use of2H2O. Its validity was tested in subjects with end-stage renal disease. Six clinically stable subjects, hemodialyzed three times weekly, ingested2H2O to a body water2H enrichment of ∼0.4%. On dialysis, body water enrichment declined to ∼0.1%. Enrichment of the α-hydrogen of plasma free alanine was also ∼0.4% before and ∼0.1% after dialysis. β-Hydrogen enrichment was ∼80-100% of α-hydrogen enrichment.2H2O was ingested to replace2H2O removed after each dialysis for 15-51 days, returning enrichment to ∼0.4%. Enrichment of alanine from plasma albumin gradually increased, with again ∼80-100% as much2H in β- as in α-hydrogens. With continued dialyses, without2H2O replacement, alanine from albumin enrichment gradually declined, whereas free alanine and water enrichments were negligible. The fractional albumin synthesis rate, calculated from the increase in enrichment in alanine from albumin, was 4.0 ± 0.5%/day, and from the decrease, 4.6 ± 0.2%/day. Thus body water enrichment in a subject given2H2O can be maintained constant long term. A rapid exchange, essentially complete, occurs between the hydrogens of alanine and body water. An integrated measure over a long period of albumin's synthetic rate can be estimated from both the rise in enrichment of alanine from the protein during2H2O ingestion and fall on2H2O withdrawal, while the subject's living routine is uninterrupted. Estimates are in subjects with renal disease, but the method should be applicable to estimates of protein synthetic rates in normal subjects and in other pathological states.
Involvement of SSAO-mediated deamination in adipose glucose transport and weight gain in obese diabetic KKAy mice
Authors: Yu Peter H. , Wang Michael , Fan Hui , Deng Yulin , Gubisne-Haberle Diana
Published: March 2004

Semicarbazide-sensitive amine oxidase (SSAO) is located on outer surfaces of adipocytes and endothelial and vascular smooth muscle cells. This enzyme catalyzes deamination of methylamine and aminoacetone, leading to production of toxic formaldehyde and methylglyoxal, respectively, as well as hydrogen peroxide and ammonium. Several lines of evidence suggest that increased SSAO activity is related to chronic inflammation and vascular disorders related to diabetic complications. We found that a highly potent and selective SSAO inhibitor, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine (FPFA), was capable of reducing numbers of atherosclerotic lesions as well as weight gain in obese KKAy mice fed an atherogenic diet. SSAO inhibitors cause a moderate and long-lasting hyperglycemia. Such an increase in serum glucose is a result of reduction of glucose uptake by adipocytes. SSAO-mediated deamination of endogenous methylamine substrates induces adipocyte glucose uptake and lipogenesis. Highly selective SSAO inhibitors can effectively block induced glucose uptake. The results suggest that increased SSAO-mediated deamination may be concomitantly related to obesity and vascular disorders associated with type 2 diabetes.
Regulation of vasopressin secretion by ETA and ETB receptors in compartmentalized rat hypothalamo-neurohypophysial explants
Authors: Rossi Noreen F.
Published: March 2004

The endothelins (ET) have been implicated in vasopressin (AVP) release in vivo and in vitro. The effects of ET in this system are complex, and the net AVP secretory response likely depends on a unique combination of ET isoform, ET receptor subtype, and neural locus. The purpose of these studies was to examine the role of ET receptor subtypes at hypothalamic vs. neurohypophysial sites on somatodendritic and neurohypophysial AVP secretion. Experiments were done in cultured explants of the hypothalamo-neurohypophysial system of Long Evans rats. Either the whole explant (standard) or only the hypothalamus or posterior pituitary (compartmentalized) was exposed to log dose increases (0.01-10 nM) of the agonists ET-1 (ETA selective), ET-3 (nonselective), or IRL-1620 (ETB selective) with or without selective ETA (BQ-123, 2-200 nM) or ETB (IRL-1038, 6-600 nM) receptor antagonism. In standard explants, ET-1 and ET-3 dose-dependently increased, whereas IRL-1620 decreased net AVP release. Hypothalamic ETB receptor activation increased both somatodendritic and neurohypophysial AVP release. At least one intervening synapse was involved, as tetrodotoxin blocked the response. Activation of ETA receptors at the hypothalamic level inhibited, whereas ETA receptor activation at the posterior pituitary stimulated, neurohypophysial AVP secretion. Antagonism of hypothalamic ETA receptors potentiated the stimulatory effect of ET-1 and ET-3 on neurohypophysial secretion, an effect not observed with ETB receptor-induced somatodendritic release of AVP. Thus the response of whole explants reflects the net result of both stimulatory and inhibitory inputs. The integration of these excitatory and inhibitory inputs endows the vasopressinergic system with greater plasticity in its response to physiological and pathophysiological states.
Effect of sympathetic denervation on the rate of protein synthesis in rat skeletal muscle
Authors: Navegantes Luiz Carlos C. , Resano Neusa M. Z. , Baviera Amanda M. , Migliorini Renato H. , Kettelhut Isis C.
Published: March 2004

Rates of protein synthesis were investigated in skeletal muscles from rats submitted to chemical and surgical sympathectomy. Three models of sympathetic denervation were used: 1) treatment with guanethidine (100 mg·kg-1·day-1sc); 2) lumbar sympathetic denervation (surgical excision of the second and third lumbar ganglia of the sympathetic chain, from which arises the postganglionic fibers to the skeletal muscles of rat hindlimb); and 3) adrenodemedullation. Protein synthesis was estimated in isolated soleus muscle by the rate of incorporation of [14C]tyrosine (0.1 mM, 0.05 μCi/ml) into total protein. Soleus isolated after 2 and 4 days of chemical sympathectomy or after 3 days of lumbar denervation showed a 17-20% statistically significant decrease in in vitro rates of protein synthesis. These effects were reverted by addition of 10-5M isoproterenol or epinephrine in vitro. Neither clenbuterol nor isoproterenol (10-7, 10-6, or 10-5M) in vitro affected the rate of protein synthesis in soleus from normal rats. On the other hand, clenbuterol or epinephrine (10-5M) increased by 20% the rate of protein synthesis in soleus muscles from adrenodemedullated rats and prevented its decrease in muscles from fasted rats. The data suggest that the sympathetic nervous system stimulates protein synthesis in oxidative muscles, probably through the activation of β2-adrenoceptors, especially in situations of hormonal or nutritional deficiency.
Glucose deprivation enhances targeting of GLUT1 to lipid rafts in 3T3-L1 adipocytes
Authors: Kumar Anil , Xiao Yu-Ping , Laipis Philip J. , Fletcher Bradley S. , Frost Susan C.
Published: March 2004

Glucose deprivation dramatically increases glucose transport activity in 3T3-L1 adipocytes without changing the concentration of GLUT1 in the plasma membrane (PM). Recent data suggest that subcompartments within the PM, specifically lipid rafts, may sequester selected proteins and alter their activity. To evaluate this possibility, we examined the distribution of GLUT1 in Triton X-100-soluble and -insoluble fractions. Our data show that 77% of the GLUT1 pool in PMs isolated from control 3T3-L1 adipocytes was extracted by 0.2% Triton X-100. After glucose deprivation for 12 h, only 56% of GLUT1 was extracted by detergent. In contrast, there was a twofold increase in the GLUT1 content of the detergent-resistant fraction. To evaluate whether GLUT1 interacts with a specific protein within lipid rafts, we focused on stomatin, recently shown to interact with and inhibit GLUT1 activity. Stomatin is distributed about equally between the PM and the biosynthetic compartments, and its expression is not affected by glucose deprivation. Nearly 90% of the PM pool of stomatin is in detergent-resistant lipid rafts. In normal 3T3-L1 adipocytes, we were unable to demonstrate an interaction between GLUT1 and stomatin in coimmunoprecipitation experiments. However, in stomatin-overexpressing cells, there was clear coprecipitation of stomatin with GLUT1 antibodies. Glucose deprivation increased this interaction threefold, which may reflect the increase of GLUT1 in lipid rafts. Despite this, there was little change in transport activity in glucose-deprived, stomatin-overexpressing cells vs. that in control cells. Thus GLUT1 interacts with stomatin in lipid rafts, but this interaction per se does not alter transport activity. Rather, stomatin may serve as an anchor for GLUT1 in lipid rafts, the environment of which favors activation.
Endothelin receptor antagonist reverses decreased NO system in the kidney in vivo during exercise
Authors: Maeda Seiji , Miyauchi Takashi , Iemitsu Motoyuki , Tanabe Takumi , Goto Katsutoshi , Yamaguchi Iwao , Matsuda Mitsuo
Published: March 2004

Vascular endothelial cells produce endothelin (ET)-1, a potent vasoconstrictor peptide, and nitric oxide (NO), a potent vasodilator substance. There are interactions between ET-1 and NO. Exercise results in a marked decrease in renal blood flow. We previously reported that exercise causes an increase of ET-1 production in the kidney, whereas production of NO in the kidney is decreased. Furthermore, we recently revealed that the magnitude of decrease in blood flow to the kidney during exercise was significantly attenuated by the administration of the endothelin-A (ETA) receptor antagonist, strongly suggesting that endogenously increased ET-1 participates in the decrease of blood flow in the kidney during exercise. Because it was demonstrated that ET-1 depresses NO synthase (NOS) activity of cultured cells in vitro, we hypothesized that an increase of ET-1 production in kidney during exercise contributes to a decrease of NO production in kidney in vivo. We studied whether administration of the ETA receptor antagonist attenuates the decreases of NOS activity and NO production in the kidney during exercise. Rats performed treadmill running for 30 min after pretreatment with an ETA receptor antagonist (TA-0201, 0.5 mg/kg; TA-0201-treated exercise group) or vehicle (vehicle-treated exercise group). Control rats remained at rest (vehicle-treated sedentary group). Blood flow in the kidney was decreased by this exercise, but the magnitude of the decrease after pretreatment with TA-0201 was significantly smaller than that after pretreatment with vehicle. NOS activity in kidney was significantly lower in the vehicle-treated exercise group than in the vehicle-treated sedentary group, whereas that in the TA-0201-treated exercise group was significantly higher than that in the vehicle-treated exercise group. Expressions of endothelial NOS protein and NOx, the stable end product of NO, i.e., nitrite/nitrate, concentration in the kidney were significantly lower in the vehicle-treated exercise group than in the vehicle-treated sedentary group, whereas those in the TA-0201-treated exercise group were significantly higher than those in the vehicle-treated exercise group. The data suggest that increased ET-1 production in the kidney during exercise contributes to the decreases of NOS activity and NO production. Therefore, the present study provides a possibility that the exercise-induced increase in production of ET-1 in the kidney causes a decrease in blood flow in the kidney through two pathways, i.e., vasoconstrictive action and the action of attenuating NO production.
Retention of estradiol negative feedback relationship to LH predicts ovulation in response to caloric restriction and weight loss in obese patients with polycystic ovary syndrome
Authors: van Dam Eveline W. C. M. , Roelfsema Ferdinand , Veldhuis Johannes D. , Hogendoorn Simone , Westenberg Jos , Helmerhorst Frans M. , Frölich Marijke , Krans H. Michiel J. , Meinders A. Edo , Pijl Hanno
Published: March 2004

The present study tests the hypothesis that specific endocrine, metabolic, and anthropometric features distinguish obese women with polycystic ovary syndrome (PCOS) who resume ovulation in response to calorie restriction and weight loss from those who do not. Fifteen obese (body mass index 39 ± 7 kg/m2) hyperandrogenemic oligoovulatory patients undertook a very low calorie diet (VLCD), wherein each lost ≥10% of body weight over a mean of 6.25 mo. Body fat distribution was quantitated by magnetic resonance imaging. Hormones were measured in the morning at baseline, after 1 wk of VLCD, and after 10% weight loss. To monitor LH release, blood was sampled for 24 h at 10-min intervals before intervention and after 7 days of VLCD. Responders were defined a priori as individuals exhibiting two or more ovulatory cycles in the course of intervention, as corroborated by serum progesterone concentrations ≥18 nmol/l followed by vaginal bleeding. At baseline, responders had a higher sex hormone-binding globulin (SHBG) concentration but were otherwise indistinguishable from nonresponders. Body weight, the size of body fat depots, and plasma insulin levels declined to a similar extent in responders and nonresponders. Also, SHBG increased, and the free testosterone index decreased comparably. However, responders exhibited a significant decline of circulating estradiol concentrations (from 191 ± 82 to 158 ± 77 pmol/l, means ± SD, P = 0.037) and a concurrent increase in LH secretion (from 104 ± 42 to 140 ± 5 U·l-1·day-1, P = 0.006) in response to 7 days of VLCD, whereas neither parameter changed significantly in nonresponders. We infer that evidence of retention of estradiol-dependent negative feedback on LH secretion may forecast follicle maturation and ovulation in obese patients with PCOS under dietary restriction.
Exercise augments the acute anabolic effects of intradialytic parenteral nutrition in chronic hemodialysis patients
Authors: Pupim Lara B. , Flakoll Paul J. , Levenhagen Deanna K. , Ikizler T. Alp
Published: March 2004

Decreased dietary protein intake and hemodialysis (HD)-associated protein catabolism are among several factors that predispose chronic hemodialysis (CHD) patients to uremic malnutrition and associated muscle wasting. Intradialytic parenteral nutrition (IDPN) acutely reverses the net negative whole body and forearm muscle protein balances observed during the HD procedure. Exercise has been shown to improve muscle protein homeostasis, especially if performed with adequately available intramuscular amino acids. We hypothesized that exercise performance would provide additive anabolic effects to the beneficial effects of IDPN. We studied six CHD patients at two separate HD sessions: 1) IDPN administration only and 2) IDPN + exercise. Patients were studied 2 h before, during, and 2 h after an HD session by use of a primed constant infusion of l-[1-13C]leucine and l-[ ring-2H5] phenylalanine. Exercise combined with IDPN promoted additive twofold increases in forearm muscle essential amino acid uptake (455 ± 105 vs. 229 ± 38 nmol·100 ml-1·min-1, P < 0.05) and net muscle protein accretion (125 ± 37 vs. 56 ± 30 μg·100 ml-1·min-1, P < 0.05) during HD compared with IDPN alone. Measurements of whole body protein homeostasis and energy expenditure were not altered by exercise treatment. In conclusion, exercise in the presence of adequate nutritional supplementation has potential as a therapeutic intervention to blunt the loss of muscle mass in CHD patients.
Involvement of Sp1 and SREBP-1a in transcriptional activation of the LDL receptor gene by insulin and LH in cultured porcine granulosa-luteal cells
Authors: Sekar Natesampillai , Veldhuis Johannes D.
Published: March 2004

Luteinizing hormone (LH) and insulin stimulate transcriptional activity of the porcine low-density lipoprotein (LDL) receptor (LDLR) promoter supra-additively in primary cultures of granulosa-luteal cells. The mechanistic basis of this bihormonal interaction is unknown. The pig LDLR gene promoter includes three putative Sp1/Sp3-binding sites and one sterol response element (SRE) site 5′ upstream to the transcriptional start site. To assess the role of SRE-binding protein (SREBP) in LDLR gene regulation, swine granulosa-luteal cells were cotransfected with CMV/SREBP-1a or SREBP-2 and the pLDLR1076/luc promoter. SREBP-1a and SREBP-2 stimulated LDLR gene transcription eight- and fourfold, respectively. LH alone augmented stimulation by SREBP-1 twofold. Conversely, cotransfection of a dominant-negative mutant form of SREBP-1a repressed basal and hormonally stimulated LDLR promoter activity by >80% ( P < 0.01). Mutation of the SRE −167 ATCACCCCATG −157 to −167 ATCACCgCATG −157 bp decreased basal expression by 50% and LH + insulin- and LH + IGF-I-stimulated transcriptional activity by 80% and >90%, respectively (both P < 0.01). Mutations within each of the three flanking putative Sp1/Sp3 sites at −216/−211, −201/−196, and −151/−146 bp in the LDLR gene promoter also reduced basal activity (by >85%) and hormonal responsiveness (>95%, P < 0.05). EMSA confirmed that presumptive SRE-1 and Sp1/Sp3 elements bind respective peptides. Mithramycin, an inhibitor of Sp1/Sp3 protein(s) binding, blocked hormonally induced LDLR promoter expression by 80%. Basal transcription and supra-additive stimulation of porcine LDLR gene transcription by LH and insulin in granulosa-luteal cells require SREBP-1a and Sp1/Sp3-binding elements.
Branched-chain amino acids increase p70S6k phosphorylation in human skeletal muscle after resistance exercise
Authors: Karlsson Håkan K. R. , Nilsson Per-Anders , Nilsson Johnny , Chibalin Alexander V. , Zierath Juleen R. , Blomstrand Eva
Published: March 2004

The aim of the study was to investigate the effect of resistance exercise alone or in combination with oral intake of branched-chain amino acids (BCAA) on phosphorylation of the 70-kDa S6 protein kinase (p70S6k) and mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK1/2), and p38 MAPK in skeletal muscle. Seven male subjects performed one session of quadriceps muscle resistance training (4 × 10 repetitions at 80% of one repetition maximum) on two occasions. In a randomized order, double-blind, crossover test, subjects ingested a solution of BCAA or placebo during and after exercise. Ingestion of BCAA increased plasma concentrations of isoleucine, leucine, and valine during exercise and throughout recovery after exercise (2 h postexercise), whereas no change was noted after the placebo trial. Resistance exercise led to a robust increase in p70S6k phosphorylation at Ser424 and/or Thr421, which persisted 1 and 2 h after exercise. BCAA ingestion further enhanced p70S6k phosphorylation 3.5-fold during recovery. p70S6k phosphorylation at Thr389 was unaltered directly after resistance exercise. However, during recovery, Thr389 phosphorylation was profoundly increased, but only during the BCAA trial. Furthermore, phosphorylation of the ribosomal protein S6 was also increased in the recovery period only during the BCAA trial. Exercise led to a marked increase in ERK1/2 and p38 MAPK phosphorylation, which was completely suppressed upon recovery and unaltered by BCAA. In conclusion, BCAA, ingested during and after resistance exercise, mediate signal transduction through p70S6k in skeletal muscle.
Coordinated increase in albumin, fibrinogen, and muscle protein synthesis during hemodialysis: role of cytokines
Authors: Raj Dominic S. C. , Dominic Elizabeth A. , Wolfe Robert , Shah Vallabh O. , Bankhurst Arthur , Zager Philip G. , Ferrando Arny
Published: March 2004

Serum albumin, fibrinogen levels, and lean body mass are important predictors of outcome in end-stage renal disease (ESRD). We estimated the fractional synthesis rates of albumin (FSR-A), fibrinogen (FSR-F), and muscle protein (FSR-M) in nine ESRD patients and eight controls, using primed constant infusion of l-[ ring-13C6]phenylalanine. Cytokine profile and arteriovenous balance of amino acids were also measured. ESRD patients were studied before (Pre-HD) and during hemodialysis (HD). Plasma IL-6, IL-10, and C-reactive protein increased significantly during HD. Despite a decrease in the delivery of amino acids to the leg, the outflow of the amino acids increased during HD. The net balance of amino acids became more negative during HD, indicating release from the muscle. HD increased leg muscle protein synthesis (45%) and catabolism (108%) but decreased whole body proteolysis (15%). FSR-A during HD (9.7 ± 0.9%/day) was higher than pre-HD (6.5 ± 0.9%/day) and controls (5.8 ± 0.5%/day, P < 0.01). FSR-F increased during HD (19.7 ± 2.6%/day vs. 11.8 ± 0.6%/day, P < 0.01), but it was not significantly different from that of controls (14.4 ± 1.4%/day). FSR-M intradialysis (1.77 ± 0.19%/day) was higher than pre-HD (1.21 ± 0.25%/day) and controls (1.30 ± 0.32%/day, P < 0.001). Pre-HD FSR-A, FSR-F, and FSR-M values were comparable to those of controls. There was a significant and positive correlation between plasma IL-6 and the FSRs. Thus, in ESRD patients without metabolic acidosis, the fractional synthesis rates of albumin, fibrinogen, and muscle protein are not decreased pre-HD. However, HD increases the synthesis of albumin, fibrinogen, and muscle protein. The coordinated increase in the FSRs is facilitated by constant delivery of amino acids derived from the muscle catabolism and intradialytic increase in IL-6.
Islet cell engraftment and control of diabetes in rats after transplantation of pig pancreatic anlagen
Authors: Rogers Sharon A. , Chen Feng , Talcott Mike , Hammerman Marc R.
Published: March 2004

The insufficient supply of tissue, loss posttransplantation, and limited potential for expansion of β-cells restrict the use of islet allotransplantation for diabetes. A way to overcome the supply and expansion problems is to xenotransplant embryonic tissue. We have shown that whole rat pancreatic anlagen isotransplanted into the omentum of rats, or xenotransplanted into costimulatory blocked mice, undergo growth and differentiate into islets surrounded by stoma without exocrine tissue. Isotransplants normalize glucose tolerance in diabetic hosts. Here, we show that embryonic day 29 porcine pancreas transplanted into the omentum of adult diabetic rats undergoes endocrine tissue differentiation over 20 wk and normalizes body weights and glucose tolerance. Unlike rat-to-rodent transplants, individual α- and β-cells engraft without a stromal component, and no immunosuppression is required for pig-to-rat transplants. Herein is described a novel means to effect the xenotransplantation of individual islet cells across a highly disparate barrier.
Effect of sex on counterregulatory responses to exercise after antecedent hypoglycemia in type 1 diabetes
Authors: Galassetti Pietro , Tate Donna , Neill Ray A. , Morrey Sachiko , Wasserman David H. , Davis Stephen N.
Published: March 2004

A marked sexual dimorphism exists in healthy individuals in the pattern of blunted neuroendocrine and metabolic responses following antecedent stress. It is unknown whether significant sex-related counterregulatory differences occur during prolonged moderate exercise after antecedent hypoglycemia in type 1 diabetes mellitus (T1DM). Fourteen patients with T1DM (7 women and 7 men) were studied during 90 min of euglycemic exercise at 50% maximal O2consumption after two 2-h episodes of previous-day euglycemia (5.0 mmol/l) or hypoglycemia of 2.9 mmol/l. Men and women were matched for age, glycemic control, duration of diabetes, and exercise fitness and had no history or evidence of autonomic neuropathy. Exercise was performed during constant “basal” intravenous infusion of regular insulin (1 U/h) and a 20% dextrose infusion, as needed to maintain euglycemia. Plasma glucose and insulin levels were equivalent in men and women during all exercise and glucose clamp studies. Antecedent hypoglycemia produced a relatively greater ( P < 0.05) reduction of glucagon, epinephrine, norepinephrine, growth hormone, and metabolic (glucose kinetics) responses in men compared with women during next-day exercise. After antecedent hypoglycemia, endogenous glucose production (EGP) was significantly reduced in men only, paralleling a reduction in the glucagon-to-insulin ratio and catecholamine responses. In conclusion, a marked sexual dimorphism exists in a wide spectrum of blunted counterregulatory responses to exercise in T1DM after prior hypoglycemia. Key neuroendocrine (glucagon, catecholamines) and metabolic (EGP) homeostatic responses were better preserved during exercise in T1DM women after antecedent hypoglycemia. Preserved counterregulatory responses during exercise in T1DM women may confer greater protection against hypoglycemia than in men with T1DM.
Ten categories of statistical errors: a guide for research in endocrinology and metabolism
Authors: Holmes Tyson H.
Published: March 2004

A simple framework is introduced that defines ten categories of statistical errors on the basis of type of error, bias or imprecision, and source: sampling, measurement, estimation, hypothesis testing, and reporting. Each of these ten categories is illustrated with examples pertinent to research and publication in the disciplines of endocrinology and metabolism. Some suggested remedies are discussed, where appropriate. A review of recent issues of American Journal of Physiology: Endocrinology and Metabolism and of Endocrinology finds that very small sample sizes may be the most prevalent cause of statistical error in this literature.
Rosiglitazone prevents the impairment of human islet function induced by fatty acids: evidence for a role of PPARγ2 in the modulation of insulin secretion
Authors: Lupi R. , Del Guerra S. , Marselli L. , Bugliani M. , Boggi U. , Mosca F. , Marchetti P. , Del Prato S.
Published: March 2004

Peroxisome proliferator-activated receptors (PPARs) are a subgroup of the superfamily of nuclear receptors, with three distinct main types: α, β and γ (subdivided into γ1 and γ2). Recently, the presence of PPARγ has been reported in human islets. Whether other PPAR types can be found in human islets, how islet PPARγ mRNA expression is regulated by the metabolic milieu, their role in insulin secretion, and the effects of a PPARγ agonist are not known. In this study, human pancreatic islets were prepared by collagenase digestion and density gradient purification from nonobese adult donors. The presence of PPAR mRNAs was assessed by RT-PCR, and the effect was evaluated of exposure for up to 24 h to either 22.2 mmol/l glucose and/or 0.25, 0.5, or 1.0 mmol/l long-chain fatty acid mixture (oleate to palmitate, 2:1). PPARβ and, to a greater extent, total PPARγ and PPARγ2 mRNAs were expressed in human islets, whereas PPARα mRNA was not detected. Compared with human adipose tissue, PPARγ mRNA was expressed at lower levels in the islets, and PPARβ at similar levels. The expression of PPARγ2 mRNA was not affected by exposure to 22.2 mmol/l glucose, whereas it decreased markedly and time dependently after exposure to progressively higher free fatty acids (FFA). This latter effect was not affected by the concomitant presence of high glucose. Exposure to FFA caused inhibition of insulin mRNA expression, glucose-stimulated insulin release, and reduction of islet insulin content. The PPARγ agonists rosiglitazone and 15-deoxy-Δ-12,14prostaglandin J2 prevented the cytostatic effect of FFA as well as the FFA-induced changes of PPAR and insulin mRNA expression. In conclusion, this study shows that PPARγ mRNA is expressed in human pancreatic islets, with predominance of PPARγ2; exposure to FFA downregulates PPARγ2 and insulin mRNA expression and inhibits glucose-stimulated insulin secretion; exposure to PPARγ agonists can prevent these effects.
Escherichia coliLPS induces heat shock protein 25 in intestinal epithelial cells through MAP kinase activation
Authors: Kojima Keishi , Musch Mark W. , Ropeleski Mark J. , Boone David L. , Ma Averil , Chang Eugene B.
Published: March 2004

Protection of colonic epithelial integrity and function is critical, because compromises in mucosal functions can lead to adverse and potentially life-threatening effects. The gut flora may contribute to this protection, in part, through the sustained induction of cytoprotective heat shock proteins (HSPs) in surface colonocytes. In this study, we investigated whether Escherichia coli LPS mediates bacteria-induced HSP by using cultured young adult mouse colon (YAMC) cells, an in vitro model of the colonic epithelium. E. coli LPS led to an epithelial cell-type specific induction of HSP25 in a time- and concentration-dependent manner, an effect that did not involve changes in HSP72. YAMC cells expressed the toll-like receptors (TLR)2 and TLR4 but not the costimulatory CD14 molecule. Whereas LPS stimulated both the p38 and ERK1/2 but not the stress-activated protein kinase/c-Jun NH2-terminal kinase, signaling pathways in the YAMC cells, all three were stimulated in RAW macrophage cells (in which no LPS-induced HSP25 expression was observed). The p38 inhibitor SB-203580 and the MAP kinase kinase-1 inhibitor PD-98059 inhibited HSP25 induction by LPS. LPS treatment also conferred protection against actin depolymerization induced by the oxidant monochloramine. The HSP25 dependence of the LPS protective effect was outlined in inhibitor studies and through adenovirus-mediated overexpression of HSP25. In conclusion, LPS may be an important mediator of enteric bacteria-induced expression of intestinal epithelial HSP25, an effect that may contribute to filamentous actin stabilization under physiological as well as pathophysiological conditions and thus protection of colonic epithelial integrity.
Linoleic acid induces interleukin-8 production by Crohn's human intestinal smooth muscle cells via arachidonic acid metabolites
Authors: Alzoghaibi Mohammad A. , Walsh Scott W. , Willey Amy , Yager Dorne R. , Fowler Alpha A. , Graham Martin F.
Published: March 2004

Previously we reported that linoleic acid (LA), but not oleic acid, caused a marked increase in the secretion of IL-8 by Crohn's human intestinal smooth muscle (HISM) cells. Antioxidants inhibited this response, implicating a role for oxidative stress and NF-κB, a transcription factor for IL-8 that is activated by oxidative stress. In this study, we examined two mechanisms whereby LA, the dietary precursor for arachidonic acid (AA), could increase the production of IL-8 via activation of AA pathways: 1) by generation of reactive oxygen species by the AA-pathway enzymes to activate NF-κB or 2) by AA metabolites. Normal and Crohn's HISM cells were exposed to LA, oxidizing solution (Ox), or oxidizing solution enriched with LA (OxLA). Exposure of cells to Ox or OxLA induced oxidative stress as determined by thiobarbituric acid reactive substances. In normal cells, Ox but not LA activated NF-κB as determined by transfection experiments and Western blot. In Crohn's cells, NF-κB was spontaneously activated and was not further activated by Ox or LA. In contrast, TNF-α markedly increased activation of NF-κB in both normal and Crohn's cells. These results indicated that LA did not increase IL-8 by activating NF-κB, so we evaluated the second mechanism of an effect of AA metabolites. In normal cells, OxLA, but not LA, markedly stimulated IL-8, whereas in Crohn's cells, both OxLA and LA stimulated IL-8. OxLA, also stimulated production of AA metabolites leukotriene B4(LTB4), PGE2, and thromboxane B2(TXB2) by normal and Crohn's cells. To determine whether AA metabolites mediated the IL-8 response, cells were treated with OxLA plus indomethacin (Indo), a cyclooxygenase inhibitor, and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor. Both Indo and NDGA blocked the IL-8 response to OxLA. To determine more specifically a role for AA metabolites, AA was used. Similar to OxLA, OxAA stimulated production of IL-8 and AA metabolites. Pinane thromboxane, a selective thromboxane synthase inhibitor and receptor blocker, inhibited OxAA stimulation of TXB2and IL-8 in a dose-response manner. MK886, a selective 5-lipoxygenase inhibitor, inhibited OxAA stimulation of LTB4and IL-8 also in a dose-response manner. Analysis of specific gene products by RT-PCR demonstrated that HISM cells expressed receptors for both thromboxane and LTB4. We conclude that AA metabolites mediated the IL-8 response to LA in HISM cells. Both cyclooxygenase and lipoxygenase pathways were involved. LA did not increase IL-8 by activating NF-κB, but NF-κB appeared to be involved, because LA increased IL-8 only in situations where NF-κB was activated, either spontaneously in Crohn's cells or by Ox in normal cells. We speculate that AA metabolites increased IL-8 production by enhancing NF-κB-dependent transcription of IL-8.
Peroxisome proliferator-activated receptor-α regulates postischemic liver injury
Authors: Okaya Tomohisa , Lentsch Alex B.
Published: March 2004

Peroxisome proliferator-activated receptor-α (PPARα) is a transcription factor that in some in vitro systems has been linked with downregulation of proinflammatory mediators, thus implicating a potential role for PPARα in the regulation of inflammatory processes. Hepatic ischemia-reperfusion injury is characterized by an intense acute inflammatory response that is dependent on a number of proinflammatory mediators. PPARα is abundantly expressed in hepatic parenchymal cells but not in Kupffer cells. This study examined whether PPARα is involved in regulation of the hepatic inflammatory response to ischemia-reperfusion. Mice nullizygous for PPARα had significantly greater liver injury than did their wild-type counterparts. Consistent with these findings, C57BL/6 mice treated with the PPARα agonist, WY-14643, had significantly less liver injury than mice receiving vehicle. PPARα-knockout mice also had greatly augmented liver neutrophil accumulation and modest increases in activation of the transcription factors NF-κB and activator protein-1. However, these effects were not associated with increased expression of proinflammatory cytokines or chemokines. In addition, PPARα-knockout mice expressed far less inducible nitric oxide synthase in liver than did wild-type mice after ischemia-reperfusion. Finally, treatment of cultured murine hepatocytes with WY-14643, a specific agonist of PPARα, protected cells against oxidant-induced injury. The data suggest that PPARα is an important regulator of the hepatic inflammatory response to ischemia-reperfusion in a manner that is independent of proinflammatory cytokines.
Immunoblockade of PSGL-1 attenuates established experimental murine colitis by reduction of leukocyte rolling
Authors: Rijcken Emile M. , Laukoetter Mike G. , Anthoni Christoph , Meier Stephanie , Mennigen Rudolf , Spiegel Hans-Ullrich , Bruewer Matthias , Senninger Norbert , Vestweber Dietmar , Krieglstein Christian F.
Published: March 2004

Recruitment of circulating leukocytes into the colonic tissue is a key feature of intestinal inflammation. P-selectin glycoprotein ligand-1 (PSGL-1) and very late antigen-4 (VLA-4) are expressed on leukocytes and play an important role in leukocyte-endothelial cell adhesive interactions. We examined the effects of immunoneutralization of PSGL-1 and VLA-4 on leukocyte recruitment in vivo in the development and treatment of experimental colitis. Chronic colitis was induced in balb/c mice by oral administration of dextran sodium sulfate (DSS). Monoclonal antibodies 2PH1 (anti-PSGL-1) and PS/2 (anti-VLA-4) or the combination of both were injected intravenously, and leukocyte adhesion was observed for 60 min in colonic submucosal venules by intravital microscopy (IVM) under isoflurane/N2O anesthesia. In addition, mice with established colitis were treated by daily intraperitoneal injections of 2PH1, PS/2, or the combination of both over 5 days. Disease activity index (DAI), histology, and myeloperoxidase (MPO) levels were compared with sham-treated DSS controls. We found that 2PH1 reduced the number of rolling leukocytes (148.7 ± 29.8 vs. 36.9 ± 8.7/0.01 mm2/30 s, P < 0.05), whereas leukocyte velocity was increased (24.0 ± 3.6 vs. 127.8 ± 11.7 μm/s, P < 0.05). PS/2 reduced leukocyte rolling to a lesser extent. Leukocyte firm adhesion was not influenced by 2PH1 but was strongly reduced by PS/2 (24.1 ± 2 vs. 4.4 ± 0.9/0.01 mm2/30 s, P < 0.05). Combined application did not cause additional effects on leukocyte adhesion. Treatment of chronic colitis with 2PH1 or PS/2 reduced DAI, mucosal injury, and MPO levels significantly. Combined treatment led to a significantly better reduction of DAI (0.4 ± 0.1 vs. 2.1 ± 0.2 points) and histology (9.7 ± 0.9 vs. 21.4 ± 4.6 points). In conclusion, PSGL-1 and VLA-4 play an important role for leukocyte recruitment during intestinal inflammation. Therapeutic strategies designed to disrupt interactions mediated by PSGL-1 and/or VLA-4 may prove beneficial in treatment of chronic colitis.