bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of infection on glucose-dependent liver glucose uptake during TPN: interaction with insulin
Authors: Chen Sheng-Song , Donmoyer Christine M. , Pearson David A. , Poole Adrian , Zhang Yiqun , Lacy D. Brooks , McGuinness Owen P.
Published: January 2004

Chronic total parenteral nutrition (TPN) markedly augments net hepatic glucose uptake (NHGU). This adaptive increase is impaired by an infection despite accompanying hyperinsulinemia. In the nonadapted state, NHGU is dependent on the prevailing glucose levels. Our aims were to determine whether the adaptation to TPN alters the glucose dependence of NHGU, whether infection impairs this dependence, and whether insulin modulates the glucose dependence of NHGU during infection. Chronically catheterized dogs received TPN for 5 days. On day 3 of TPN, dogs received either a bacterial fibrin clot to induce a nonlethal infection (INF, n = 9) or a sterile fibrin clot (Sham, n = 6). Forty-two hours after clot implantation, somatostatin was infused. In Sham, insulin and glucagon were infused to match the level seen in Sham (9 ± 1 μU/ml and 23 ± 4 pg/ml, respectively). In infected animals, either insulin and glucagon were infused to match the levels seen in infection (25 ± 2 μU/ml and 101 ± 15 pg/ml; INF-HI; n = 5) or insulin was replaced to match the lower levels seen in Sham (13 ± 2 μU/ml), whereas glucagon was kept elevated (97 ± 9 pg/ml; INF-LO; n = 4). Then a four-step (90 min each) hyperglycemic (120, 150, 200, or 250 mg/dl) clamp was performed. NHGU increased at each glucose step in Sham (from 3.6 ± 0.6 to 5.4 ± 0.7 to 8.9 ± 0.9 to 12.1 ± 1.1 mg·kg–1·min–1); the slope of the relationship between glucose levels and NHGU (i.e., glucose dependence) was higher than that seen in nonadapted animals. Infection impaired glucose-dependent NHGU in both INF-HI (1.3 ± 0.4 to 2.9 ± 0.5 to 5.5 ± 1.0 to 7.7 ± 1.6 mg·kg–1·min–1) and INF-LO (0.5 ± 0.7 to 2.2 ± 0.6 to 4.2 ± 1.0 to 5.8 ± 0.8 mg·kg–1·min–1). In summary, TPN augments glucose-dependent NHGU, the presence of infection decreases glucose-dependent NHGU, and the accompanying hyperinsulinemia associated with infection does not sustain the glucose dependence of NHGU.
Endurance training partially reverses dietary-induced leptin resistance in rodent skeletal muscle
Authors: Steinberg Gregory R. , Smith Angela C. , Wormald Sam , Malenfant Patrick , Collier Cheryl , Dyck David J.
Published: January 2004

Leptin acutely stimulates skeletal muscle fatty acid (FA) metabolism in lean rodents and humans. This stimulatory effect is eliminated following the feeding of high-fat diets in rodents as well as in obese humans. The mechanism(s) responsible for the development of skeletal muscle leptin resistance is unknown; however, a role for increased suppressor of cytokine signaling-3 (SOCS3) inhibition of the leptin receptor has been demonstrated in other rodent tissues. Furthermore, whether exercise intervention is an effective strategy to prevent or attenuate the development of skeletal muscle leptin resistance has not been investigated. Toward this end, 48 Sprague-Dawley rats (175-190 g; ∼2-3 mo of age) were fed control or high-fat (60% kcal) diets for 4 wk and either remained sedentary or were treadmill trained. In control diet-fed animals that remained sedentary (CS) or were endurance trained (CT), leptin stimulated FA oxidation (CS +32 ± 15%, CT +30 ± 17%; P < 0.05), suppressed triacylglycerol (TAG) esterification (CS -17 ± 7%, CT -24 ± 8%; P < 0.05), and reduced the esterification-to-oxidation ratio (CS -19 ± 13%, CT -29 ± 10%; P < 0.001) in soleus muscle. High-fat feeding induced leptin resistance in the soleus of sedentary rats (FS), whereas endurance exercise training (FT) restored the ability of leptin to suppress TAG esterification (-19 ± 9%, P = 0.038). Training did not completely restore the ability of leptin to stimulate FA oxidation. High-fat diets stimulated SOCS3 mRNA expression irrespective of training status (FS +451 ± 120%, P = 0.024; FT +381 ± 141%, P = 0.023). Thus the development of skeletal muscle leptin resistance appears to involve an increase in SOCS3 mRNA expression. Endurance training was generally effective in preventing the development of leptin resistance, although this did not appear to require a decrease in SOCS3 expression. Future studies should examine changes in the actual protein content of SOCS3 in muscle and establish whether aerobic exercise is also effective in treating leptin resistance in humans.
Effect of high-intensity intermittent training on lactate and H+ release from human skeletal muscle
Authors: Juel Carsten , Klarskov Christina , Nielsen Jens Jung , Krustrup Peter , Mohr Magni , Bangsbo Jens
Published: January 2004

The study investigated the effect of training on lactate and H+ release from human skeletal muscle during one-legged knee-extensor exercise. Six subjects were tested after 7–8 wk of training (fifteen 1-min bouts at ∼150% of thigh maximal O2 uptake per day). Blood samples, blood flow, and muscle biopsies were obtained during and after a 30-W exercise bout and an incremental test to exhaustion of both trained (T) and untrained (UT) legs. Blood flow was 16% higher in the T than in the UT leg. In the 30-W test, venous lactate and lactate release were lower in the T compared with the UT leg. In the incremental test, time to fatigue was 10.6 ± 0.7 and 8.2 ± 0.7 min, respectively, in the T and UT legs ( P < 0.05). At exhaustion, venous blood lactate was 10.7 ± 0.4 and 8.0 ± 0.9 mmol/l in T and UT legs ( P < 0.05), respectively, and lactate release was 19.4 ± 3.6 and 10.6 ± 2.0 mmol/min ( P < 0.05). H+ release at exhaustion was higher in the T than in the UT leg. Muscle lactate content was 59.0 ± 15.1 and 96.5 ± 14.5 mmol/kg dry wt in the T and UT legs, and muscle pH was 6.82 ± 0.05 and 6.69 ± 0.04 in the T and UT legs ( P = 0.06). The membrane contents of the monocarboxylate transporters MCT1 and MCT4 and the Na+/H+ exchanger were 115 ± 5 ( P < 0.05), 111 ± 11, and 116 ± 6% ( P < 0.05), respectively, in the T compared with the UT leg. The reason for the training-induced increase in peak lactate and H+ release during exercise is a combination of an increased density of the lactate and H+ transporting systems, an improved blood flow and blood flow distribution, and an increased systemic lactate and H+ clearance.
Distributed control of glucose uptake by working muscles of conscious mice: roles of transport and phosphorylation
Authors: Fueger Patrick T. , Bracy Deanna P. , Malabanan Carlo M. , Pencek R. Richard , Wasserman David H.
Published: January 2004

Muscle glucose uptake (MGU) is determined by glucose delivery, transport, and phosphorylation. C57Bl/6J mice overexpressing GLUT4, hexokinase II (HK II), or both were used to determine the barriers to MGU. A carotid artery and jugular vein were catheterized for arterial blood sampling and venous infusions. Experiments were conducted in conscious mice ∼7 days after surgery. 2-Deoxy-[3H]glucose was administered during rest or treadmill exercise to calculate glucose concentration-dependent (Rg) and -independent (Kg) indexes of MGU. Compared with wild-type controls, GLUT4-overexpressing mice had lowered fasting glycemia (165 ± 6 vs. 115 ± 6 mg/dl) and increased Rgby 230 and 166% in the gastrocnemius and superficial vastus lateralis (SVL) muscles under sedentary conditions. GLUT4 overexpression was not able to augment exercise-stimulated Rgor Kg. Whereas HK II overexpression had no effect on fasting glycemia (170 ± 6 mg/dl) or sedentary Rg, it increased exercise-stimulated Rgby 82, 60, and 169% in soleus, gastrocnemius, and SVL muscles, respectively. Combined GLUT4 and HK II overexpression lowered fasting glycemia (106 ± 6 mg/dl), increased nonesterified fatty acids, and increased sedentary Rg. Combined GLUT4 and HK II overexpression did not enhance exercise-stimulated Rgcompared with HK II-overexpressing mice because of the reduced glucose concentration. GLUT4 combined with HK II overexpression resulted in a marked increase in exercise-stimulated Kg. In conclusion, control of MGU shifts from membrane transport at rest to phosphorylation during exercise. Glucose transport is not normally a significant barrier during exercise. However, when the phosphorylation barrier is lowered by HK II overexpression, glucose transport becomes a key site of control for regulating MGU during exercise.
AMPK activity and isoform protein expression are similar in muscle of obese subjects with and without type 2 diabetes
Authors: Højlund Kurt , Mustard Kirsty J. , Stæhr Peter , Hardie D. Grahame , Beck-Nielsen Henning , Richter Erik A. , Wojtaszewski Jørgen F. P.
Published: January 2004

Acute or chronic activation of AMP-activated protein kinase (AMPK) increases insulin sensitivity. Conversely, reduced expression and/or function of AMPK might play a role in insulin resistance in type 2 diabetes. Thus protein expression of the seven subunit isoforms of AMPK and activities and/or phosphorylation of AMPK and acetyl-CoA carboxylase-β (ACCβ) was measured in skeletal muscle from obese type 2 diabetic and well-matched control subjects during euglycemic-hyperinsulinemic clamps. Protein expression of all AMPK subunit isoforms (α1, α2, β1, β2, γ1, γ2, and γ3) in muscle of obese type 2 diabetic subjects was similar to that of control subjects. In addition, α1- and α2-associated activities of AMPK, phosphorylation of α-AMPK subunits at Thr172, and phosphorylation of ACCβ at Ser221showed no difference between the two groups and were not regulated by physiological concentrations of insulin. These data suggest that impaired insulin action on glycogen synthesis and lipid oxidation in skeletal muscle of obese type 2 diabetic subjects is unlikely to involve changes in AMPK expression and activity.
Pioglitazone preserves pancreatic islet structure and insulin secretory function in three murine models of type 2 diabetes
Authors: Diani A. R. , Sawada Geri , Wyse Beatrice , Murray F. T. , Khan Mehmood
Published: January 2004

Thiazolidinediones may slow the progression of type 2 diabetes by preserving pancreatic β-cells. The effects of pioglitazone (PIO) on structure and function of β-cells in KKA(y), C57BL/6J ob/ob, and C57BL/KsJ db/db mice (genetic models of type 2 diabetes) were examined. ob/ob ( n = 7) and db/db ( n = 9) mice were randomly assigned to 50-125 mg·kg body wt-1·day-1of PIO in chow beginning at 6-10 wk of age. Control ob/ob ( n = 7) and db/db mice ( n = 9) were fed chow without PIO. KKA(y) mice ( n = 15) were fed PIO daily at doses of 62-144 mg·kg body wt-1·day-1. Control KKA(y) mice ( n = 10) received chow without PIO. Treatment continued until euthanasia at 14-26 wk of age. Blood was collected at baseline (before treatment) and just before euthanasia and was analyzed for glucose, glycosylated hemoglobin, and plasma insulin. Some of the splenic pancreas of each animal was resected and partially sectioned for light or electron microscopy. The remainder of the pancreas was assayed for insulin content. Compared with baseline and control groups, PIO treatment significantly reduced blood glucose and glycosylated hemoglobin levels. Plasma insulin levels decreased significantly in ob/ob mice treated with PIO. All groups treated with PIO exhibited significantly greater β-cell granulation, evidence of reduced β-cell stress, and 1.5- to 15-fold higher levels of pancreatic insulin. The data from these studies suggest that comparable effects would be expected to slow the progression of type 2 diabetes, either delaying or possibly preventing progression to an insulin-dependent state.
Effects of high altitude and water deprivation on arginine vasopressin release in men
Authors: Maresh C. M. , Kraemer W. J. , Judelson D. A. , VanHeest J. L. , Trad L. , Kulikowich J. M. , Goetz K. L. , Cymerman A. , Hamilton A. J.
Published: January 2004

High-altitude exposure changes the distribution of body water and electrolytes. Arginine vasopressin (AVP) may influence these alterations. The purpose of this study was to examine the effect of a 24-h water deprivation trial (WDT) on AVP release after differing altitude exposures. Seven healthy males (age 22 ± 1 yr, height 176 ± 2 cm, mass 75.3 ± 1.8 kg) completed three WDTs: at sea level (SL), after acute altitude exposure (2 days) to 4,300 m (AA), and after prolonged altitude exposure (20 days) to 4,300 m (PA). Body mass, standing and supine blood pressures, plasma osmolality (Posm), and plasma AVP (PAVP) were measured at 0, 12, 16, and 24 h of each WDT. Urine volume was measured at each void throughout testing. Baseline Posm increased from SL to altitude (SL 291.7 ± 0.8 mosmol/kgH2O, AA 299.6 ± 2.2 mosmol/kgH2O, PA 302.3 ± 1.5 mosmol/kgH2O, P < 0.05); however, baseline PAVP measurements were similar. Despite similar Posm values, the maximal PAVP response during the WDT (at 16 h) was greater at altitude than at SL (SL 1.7 ± 0.5 pg/ml, AA 6.4 ± 0.7 pg/ml, PA 8.7 ± 0.9 pg/ml, P < 0.05). In conclusion, hypoxia appeared to alter AVP regulation by raising the osmotic threshold and increasing AVP responsiveness above that threshold.
Tracer-derived total and folate-dependent homocysteine remethylation and synthesis rates in humans indicate that serine is the main one-carbon donor
Authors: Davis Steven R. , Stacpoole Peter W. , Williamson Jerry , Kick Lilia S. , Quinlivan Eoin P. , Coats Bonnie S. , Shane Barry , Bailey Lynn B. , Gregory Jesse F.
Published: January 2004

Hyperhomocysteinemia in humans is associated with genetic variants of several enzymes of folate and one-carbon metabolism and deficiencies of folate and vitamins B12and B6. In each case, hyperhomocysteinemia might be caused by diminished folate-dependent homocysteine remethylation, but this has not been confirmed in vivo. Because published stable isotopic tracer approaches cannot distinguish folate-dependent from folate-independent remethylation, we developed a dual-tracer procedure in which a [U-13C5]-methionine tracer is used in conjunction with a [3-13C]serine tracer to simultaneously measure rates of total and folate-dependent homocysteine remethylation. In young female subjects, plasma [U-13C4]homocysteine enrichment, a surrogate measure of intracellular [U-13C5]methionine enrichment, reached ∼90% of the plasma [U-13C5]methionine enrichment. Methionine-methyl and -carboxyl group fluxes were in the range of previous reports (∼25 and ∼17 μmol·kg–1·h–1, respectively). However, the rate of overall homocysteine remethylation (∼8 μmol·kg–1·h–1) was twice that of previous reports, which suggests a larger role for homocysteine remethylation in methionine metabolism than previously thought. By use of estimates of intracellular [3-13C]serine enrichment based on a conservative correction of plasma [3-13C]serine enrichment, serine was calculated to contribute ∼100% of the methyl groups used for total body homocysteine remethylation under the conditions of this protocol. This contribution represented only a small fraction (∼2.8%) of total serine flux. Our dual-tracer procedure is well suited to measure the effects of nutrient deficiencies, genetic polymorphisms, and other metabolic perturbations on homocysteine synthesis and total and folate-dependent homocysteine remethylation.
Protein turnover and amino acid transport kinetics in end-stage renal disease
Authors: Raj Dominic S. C. , Zager Philip , Shah Vallbh O. , Dominic Elizabeth A. , Adeniyi Oladipo , Blandon Pedro , Wolfe Robert , Ferrando Arny
Published: January 2004

Protein and amino acid metabolism is abnormal in end-stage renal disease (ESRD). Protein turnover is influenced by transmembrane amino acid transport. The effect of ESRD and hemodialysis (HD) on intracellular amino acid transport kinetics is unknown. We studied intracellular amino acid transport kinetics and protein turnover by use of stable isotopes of phenylalanine, leucine, lysine, alanine, and glutamine before and during HD in six ESRD patients. Data obtained from amino acid concentrations and enrichment in the artery, vein, and muscle compartments were used to calculate intracellular amino acid transport and muscle protein synthesis and catabolism. Fractional muscle protein synthesis (FSR) was estimated by the precursor product approach. Despite a significant decrease in the plasma concentrations of amino acids in the artery and vein during HD, the intracellular concentrations remained stable. Outward transport of the amino acids was significantly higher than the inward transport during HD. FSR increased during HD (0.0521 ± 0.0043 vs. 0.0772 ± 0.0055%/h, P < 0.01). Results derived from compartmental modeling indicated that both protein synthesis (118.3 ± 20.6 vs. 146.5 ± 20.6 nmol·min-1·100 ml leg-1, P < 0.01) and catabolism (119.8 ± 18.0 vs. 174.0 ± 14.2 nmol·min-1·100 ml leg-1, P < 0.01) increased during HD. However, the intradialytic increase in catabolism exceeded that of synthesis (57.8 ± 13.8 vs. 28.0 ± 8.5%, P < 0.05). Thus HD alters amino acid transport kinetics and increases protein turnover, with net increase in protein catabolism.
Age and aerobic exercise training effects on whole body and muscle protein metabolism
Authors: Short Kevin R. , Vittone Janet L. , Bigelow Maureen L. , Proctor David N. , Nair K. Sreekumaran
Published: January 2004

Aging in humans is associated with loss of lean body mass, but the causes are incompletely defined. Lean tissue mass and function depend on continuous rebuilding of proteins. We tested the hypotheses that whole body and mixed muscle protein metabolism declines with age in men and women and that aerobic exercise training would partly reverse this decline. Seventy-eight healthy, previously untrained men and women aged 19-87 yr were studied before and after 4 mo of bicycle training (up to 45 min at 80% peak heart rate, 3-4 days/wk) or control (flexibility) activity. At the whole body level, protein breakdown (measured as [13C]leucine and [15N]phenylalanine flux), Leu oxidation, and protein synthesis (nonoxidative Leu disposal) declined with age at a rate of 4-5% per decade ( P < 0.001). Fat-free mass was closely correlated with protein turnover and declined 3% per decade ( P < 0.001), but even after covariate adjustment for fat-free mass, the decline in protein turnover with age remained significant. There were no differences between men and women after adjustment for fat-free mass. Mixed muscle protein synthesis also declined with age 3.5% per decade ( P < 0.05). Exercise training improved aerobic capacity 9% overall ( P < 0.01), and mixed muscle protein synthesis increased 22% ( P < 0.05), with no effect of age on the training response for either variable. Fat-free mass, whole body protein turnover, and resting metabolic rate were unchanged by training. We conclude that rates of whole body and muscle protein metabolism decline with age in men and women, thus indicating that there is a progressive decline in the body's remodeling processes with aging. This study also demonstrates that aerobic exercise can enhance muscle protein synthesis irrespective of age.
Overexpression of constitutively activated glutamate dehydrogenase induces insulin secretion through enhanced glutamate oxidation
Authors: Anno Takatoshi , Uehara Shunsuke , Katagiri Hideki , Ohta Yasuharu , Ueda Kohei , Mizuguchi Hiroyuki , Moriyama Yoshinori , Oka Yoshitomo , Tanizawa Yukio
Published: January 2004

Glutamate dehydrogenase (GDH) catalyzes reversible oxidative deamination of l-glutamate to α-ketoglutarate. Enzyme activity is regulated by several allosteric effectors. Recognition of a new form of hyperinsulinemic hypoglycemia, hyperinsulinism/hyperammonemia (HI/HA) syndrome, which is caused by gain-of-function mutations in GDH, highlighted the importance of GDH in glucose homeostasis. GDH266C is a constitutively activated mutant enzyme we identified in a patient with HI/HA syndrome. By overexpressing GDH266C in MIN6 mouse insulinoma cells, we previously demonstrated unregulated elevation of GDH activity to render the cells responsive to glutamine in insulin secretion. Interestingly, at low glucose concentrations, basal insulin secretion was exaggerated in such cells. Herein, to clarify the role of GDH in the regulation of insulin secretion, we studied cellular glutamate metabolism using MIN6 cells overexpressing GDH266C (MIN6-GDH266C). Glutamine-stimulated insulin secretion was associated with increased glutamine oxidation and decreased intracellular glutamate content. Similarly, at 5 mmol/l glucose without glutamine, glutamine oxidation also increased, and glutamate content decreased with exaggerated insulin secretion. Glucose oxidation was not altered. Insulin secretion profiles from GDH266C-overexpressing isolated rat pancreatic islets were similar to those from MIN6-GDH266C, suggesting observation in MIN6 cells to be relevant in native β-cells. These results demonstrate that, upon activation, GDH oxidizes glutamate to α-ketoglutarate, thereby stimulating insulin secretion by providing the TCA cycle with a substrate. No evidence was obtained supporting the hypothesis that activated GDH produced glutamate, a recently proposed second messenger of insulin secretion, by the reverse reaction, to stimulate insulin secretion.
Impaired basal glucose effectiveness but unaltered fasting glucose release and gluconeogenesis during short-term hypercortisolemia in healthy subjects
Authors: Nielsen Michael F. , Caumo Andrea , Chandramouli Visvanathan , Schumann William C. , Cobelli Claudio , Landau Bernard R. , Vilstrup Hendrik , Rizza Robert A. , Schmitz Ole
Published: January 2004

Excess cortisol has been demonstrated to impair hepatic and extrahepatic insulin action. To determine whether glucose effectiveness and, in terms of endogenous glucose release (EGR), gluconeogenesis, also are altered by hypercortisolemia, eight healthy subjects were studied after overnight infusion with hydrocortisone or saline. Glucose effectiveness was assessed by a combined somatostatin and insulin infusion protocol to maintain insulin concentration at basal level in the presence of prandial glucose infusions. Despite elevated insulin concentrations ( P < 0.05), hypercortisolemia resulted in higher glucose ( P < 0.05) and free fatty acid concentrations ( P < 0.05). Furthermore, basal insulin concentrations were higher during hydrocortisone than during saline infusion ( P < 0.01), indicating the presence of steroid-induced insulin resistance. Postabsorptive glucose production ( P = 0.64) and the fractional contribution of gluconeogenesis to EGR ( P = 0.33) did not differ on the two study days. During the prandial glucose infusion, the integrated glycemic response above baseline was higher in the presence of hydrocortisone than during saline infusion ( P < 0.05), implying a decrease in net glucose effectiveness (4.42 ± 0.52 vs. 6.65 ± 0.83 ml·kg-1·min-1; P < 0.05). To determine whether this defect is attributable to an impaired ability of glucose to suppress glucose production, to stimulate its own uptake, or both, glucose turnover and “hot” (labeled) indexes of glucose effectiveness (GE) were calculated. Hepatic GE was lower during cortisol than during saline infusion (2.39 ± 0.24 vs. 3.82 ± 0.51 ml·kg-1·min-1; P < 0.05), indicating a defect in the ability of glucose to restrain its own production. In addition, in the presence of excess cortisol, glucose disappearance was inappropriate for the prevailing glucose concentration, implying a decrease in glucose clearance ( P < 0.05). The decrease in glucose clearance was confirmed by the higher increment in [3-3H]glucose during hydrocortisone than during saline infusion ( P < 0.05), despite the administration of identical tracer infusion rates. In conclusion, short-term hypercortisolemia in healthy individuals with normal β-cell function decreases insulin action but does not alter rates of EGR and gluconeogenesis. In addition, cortisol impairs the ability of glucose to suppress its own production, which due to accumulation of glucose in the glucose space results in impaired peripheral glucose clearance. These results suggest that cortisol excess impairs glucose tolerance by decreasing both insulin action and glucose effectiveness.
Beneficial effects of estrogen treatment in the HLA-B27 transgenic rat model of inflammatory bowel disease
Authors: Harnish Douglas C. , Albert Leo M. , Leathurby Yelena , Eckert Amy M. , Ciarletta Agnes , Kasaian Marion , Keith James C.
Published: January 2004

A well-established model of bowel inflammation is the HLA-B27 transgenic rat that exhibits a spontaneous disease phenotype resulting in chronic diarrhea caused by immune cell activation. Estrogens have previously been shown to modulate the immune system, and both estrogen receptors (ERα and ERβ) are present in the intestine and cells of the immune system. Therefore, the ability of estrogen to ameliorate disease progression in the HLA-B27 transgenic rat was determined. HLA-B27 transgenic rats with chronic diarrhea were treated with 17α-ethynyl-17β-estradiol (EE) for 5 days. EE treatment dramatically improved stool scores after only 3 days. Histological scores of the degree of ulceration, inflammatory cell infiltration, fibrosis, and lesion depth of the colon were also improved by EE treatment. Because neutrophil infiltration into the colon is involved in the development and propagation of disease, myeloperoxidase (MPO) activity was measured. MPO levels were reduced by 80% by EE treatment. Cotreatment with the pure ER antagonist ICI-182780 (ICI) blocked the effects of EE on stool character, MPO activity, and histology scores, strongly suggesting that the activity of EE is mediated through ER. Mast cell proteases can promote neutrophil infiltration, and gene expression analysis demonstrated that mast cell protease 1, 3, and 4 mRNA were all decreased in colons from estrogen-treated rats. In addition, a direct effect of estrogen on bone marrow-derived mast cell activity was demonstrated, suggesting that ER-mediated inactivation of mast cells may contribute to the improvement in the clinical sign and histological scores in this model.
Coordinated gastric and sphincter motility evoked by intravenous CCK-8 as monitored by ultrasonomicrometry in rats
Authors: Adelson David W. , Million Mulugeta , Kanamoto Koki , Palanca Tiffany , Taché Yvette
Published: January 2004

Gastric and sphincter motility evoked by intravenous injection of CCK-8 were investigated in urethane-anesthetized rats. Digital ultrasonomicrometry was used to monitor pyloric (PYL), antral (ANT), corpus (COR), and lower esophageal sphincter (LES) movements while simultaneously measuring intragastric pressure (IGP) and, in some experiments, subdiaphragmatic intraesophageal pressure (sIEP). Intracrystal distances (ICD) were measured continuously between pairs of piezoelectric crystals affixed to the serosa of PYL, ANT, COR (circular and longitudinal), and LES. Consecutive intravenous injections of CCK-8 (0.3, 1, and 3 μg/kg) at 30-min intervals caused dose-dependent simultaneous tonic contractions of PYL and ANT, LES opening, and drops in IGP with peak changes at 3 μg/kg of -17.9 ± 2.1, -7.7 ± 2.5, 6.5 ± 1.4, and -29.2 ± 3.8%, respectively, whereas intravenous saline had no effect. Rhythmic contractile activity was inhibited by CCK-8. COR responses were not significantly different from vehicle controls for most metrics, and the direction of response for circular COR varied between preparations, although not for repeated trials in a single preparation. During the LES response to CCK-8, sIEP rose in parallel with drops in IGP, indicating formation of a common cavity. Recovery of LES ICD after intravenous CCK occurred more rapidly than recovery of PYL ICD, suggesting the importance of preventing simultaneous patency of gastroesophageal and gastroduodenal passages. The CCKA receptor antagonist devazepide (500 μg/kg intravenous) inhibited motion responses evoked by intravenous CCK-8. These data revealed CCK-8-induced gastric and sphincter activity consistent with retropulsion of gastric content.
Influence of duodenal acidification on the sensorimotor function of the proximal stomach in humans
Authors: Lee Kwang-Jae , Vos Rita , Janssens Jozef , Tack Jan
Published: January 2004

Decreased acid clearance and increased exposure to acid of the duodenum have been reported in a subset of functional dyspepsia patients. However, the mechanism by which increased duodenal acid exposure may affect symptoms is unclear. The aim of the present study was to investigate the effects of duodenal acidification on proximal gastric tone and mechanosensitivity in humans. An infusion tube with a pH electrode attached was positioned in the second part of the duodenum, and a barostat bag was located in the gastric fundus. In 12 healthy subjects, fundic tone and sensitivity to distensions were assessed before and during duodenal infusion of 0.1 N hydrochloric acid or saline in a randomized, double-blind design. In 10 healthy subjects, meal-induced accommodation was measured during duodenal infusion of acid or saline. Acid infusion in the duodenum significantly increased fundic compliance and decreased fasting fundic tone. This was accompanied by a significant decrease in the pressures and the corresponding wall tensions at the thresholds for discomfort. During infusion of acid, significantly higher perception and symptom scores were obtained for the same distending pressures. The meal-induced fundic relaxation was significantly smaller during acid infusion compared with saline infusion. In conclusion, duodenal acidification induces proximal gastric relaxation, increases sensitivity to gastric distension, and inhibits gastric accommodation to a meal. Through these mechanisms, increased duodenal acid exposure may be involved in the pathogenesis of dyspeptic symptoms.
A role for guanylate cyclase C in acid-stimulated duodenal mucosal bicarbonate secretion
Authors: Rao S. P. , Sellers Z. , Crombie D. L. , Hogan D. L. , Mann E. A. , Childs D. , Keely S. , Sheil-Puopolo M. , Giannella R. A. , Barrett K. E. , Isenberg J. I. , Pratha V. S.
Published: January 2004

Luminal acidification provides the strongest physiological stimulus for duodenal [Formula: see text] secretion. Various neurohumoral mechanisms are believed to play a role in acid-stimulated [Formula: see text] secretion. Previous studies in the rat and human duodenum have shown that guanylin and Escherichia coli heat-stable toxin, both ligands of the transmembrane guanylyl cyclase receptor [guanylate cyclase C (GC-C)], are potent stimulators for duodenal [Formula: see text] secretion. We postulated that the GC-C receptor plays an important role in acid-stimulated [Formula: see text] secretion. In vivo perfusion studies performed in wild-type (WT) and GC-C knockout (KO) mice indicated that acid-stimulated duodenal [Formula: see text] secretion was significantly decreased in the GC-C KO animals compared with the WT counterparts. Pretreatment with PD-98059, an MEK inhibitor, resulted in attenuation of duodenal [Formula: see text] secretion in response to acid stimulation in the WT mice with no further effect in the KO mice. In vitro cGMP generation studies demonstrated a significant and comparable increase in cGMP levels on acid exposure in the duodenum of both WT and KO mice. In addition, a rapid, time-dependent phosphorylation of ERK was observed with acid exposure in the duodenum of WT mice, whereas a marked attenuation in ERK phosphorylation was observed in the KO animals despite equivalent levels of ERK in both groups of animals. On the basis of these studies, we conclude that transmembrane GC-C is a key mediator of acid-stimulated duodenal [Formula: see text] secretion. Furthermore, ERK phosphorylation may be an important intracellular mediator of duodenal [Formula: see text] secretion.
Ethanol differentially regulates NF-κB activation in pancreatic acinar cells through calcium and protein kinase C pathways
Authors: Gukovskaya Anna S. , Hosseini Saeed , Satoh Akihiko , Cheng Jason H. , Nam Kyung J. , Gukovsky Ilya , Pandol Stephen J.
Published: January 2004

Mechanisms of alcoholic pancreatitis remain unknown. Previously, we showed that ethanol feeding sensitizes rats to pancreatitis caused by CCK-8, at least in part, by augmenting activation of the proinflammatory transcription factor NF-κB. To elucidate the mechanism of sensitization, here we investigate the effect of ethanol on Ca2+- and PKC-mediated pathways of CCK-induced NF-κB activation using an in vitro system of rat pancreatic acini incubated with ethanol. Ethanol augmented CCK-8-induced activation of NF-κB, similar to our in vivo findings with ethanol-fed rats. In contrast, ethanol prevented NF-κB activation caused by thapsigargin, an agent that mobilizes intracellular Ca2+bypassing the receptor. Pharmacological analysis showed that NF-κB activation by thapsigargin but not by CCK-8 is mediated through the calcineurin pathway and that the inhibitory effect of ethanol on the thapsigargin-induced NF-κB activation could be through inhibiting this pathway. Ethanol augmented NF-κB activation induced by the phorbol ester PMA, a direct activator of PKC. Inhibitory analysis demonstrated that Ca2+-independent (novel and/or atypical) PKC isoforms are involved in NF-κB activation induced by both CCK-8 and PMA in cells treated and not treated with ethanol. The results indicate that ethanol differentially affects the Ca2+/calcineurin- and PKC-mediated pathways of NF-κB activation in pancreatic acinar cells. These effects may play a role in the ability of ethanol to sensitize pancreas to the inflammatory response and pancreatitis.
NHE3 inhibition activates duodenal bicarbonate secretion in the rat
Authors: Furukawa Osamu , Bi Luke C. , Guth Paul H. , Engel Eli , Hirokawa Masahiko , Kaunitz Jonathan D.
Published: January 2004

We examined the effect of inhibition of Na+/H+exchange (NHE) on duodenal bicarbonate secretion (DBS) in rats to further understand DBS regulation. DBS was measured by using the pH-stat method and by using CO2-sensitive electrodes. 5-( N,N-dimethyl)-amiloride (50 μM; DMA), a concentration that selectively inhibits the NHE isoforms NHE1 and NHE2, but not NHE3, did not affect DBS. Nevertheless, 3 mM DMA, a higher concentration that inhibits NHE1, NHE2, and NHE3, significantly increased DBS. Moreover, S1611 and S3226, both specific inhibitors of NHE3 only, or perfusion with Na+-free solutions, dose dependently increased DBS, as measured by pH-stat and CO2-sensitive electrode, without affecting intracellular pH. Coperfusion with 0.1 μM indomethacin, 0.5 mM DIDS, or 1 mM methazolamide did not affect S3226-induced DBS. Nevertheless, coperfusion with 0.1 and 0.3 mM 5-nitro-2-(3-phenylpropylamino) benzoic acid, which inhibits the cystic fibrosis transmembrane conductor regulator (CFTR), dose dependently inhibited S3226-induced DBS. In conclusion, only specific apical NHE3 inhibition increased DBS, whereas prostaglandin synthesis, [Formula: see text] cotransporter activation, or intracellular [Formula: see text] formation by carbonic anhydrase was not involved. Because NHE3 inhibition-increased DBS was inhibited by an anion channel inhibitor and because reciprocal CFTR regulation has been previously shown between NHE3 and apical membrane anion transporters, we speculate that NHE3 inhibition increased DBS by altering anion transporter function.
Anti-inflammatory effect of two isoforms of COX inH. pylori-induced gastritis in mice: possible involvement of PGE2
Authors: Tanigawa Tetsuya , Watanabe Toshio , Hamaguchi Masaki , Sasaki Eiji , Tominaga Kazunari , Fujiwara Yasuhiro , Oshitani Nobuhide , Matsumoto Takayuki , Higuchi Kazuhide , Arakawa Tetsuo
Published: January 2004

Neutrophil infiltration mediated by TNF-α is associated with various types of gastric injury, whereas PGs play a crucial role in gastric defense. We examined roles of two isoforms of cyclooxygenase (COX) and PGE2in Helicobacter pylori-induced gastritis in mice. Mice infected with H. pylori were given selective COX-1 inhibitor SC-560 (10 mg/kg), selective COX-2 inhibitor NS-398 (10 mg/kg), or nonselective COX inhibitor indomethacin (2 mg/kg) with or without 16,16-dimethyl PGE2for 1 wk. H. pylori infection increased levels of mRNA for COX-1 and -2 in gastric tissue by 1.2-fold and 3.3-fold, respectively, accompanied by a significant increase in PGE2production by gastric tissue. H. pylori infection significantly elevated MPO activity, a marker of neutrophil infiltration, and epithelial cell apoptosis in the stomach. SC-560 augmented MPO activity and epithelial cell apoptosis with associated reduction in PGE2production, whereas NS-398 had the same effects without affecting PGE2production. Inhibition of both COX-1 and -2 by indomethacin or concurrent treatment with SC-560 and NS-398 resulted in a stronger increase in MPO activity and apoptosis than inhibition of either COX-1 or -2 alone. H. pylori infection elevated TNF-α mRNA expression in the stomach, which was further increased by indomethacin. Effects of COX inhibitors on neutrophil infiltration, apoptosis, and TNF-α expression in H. pylori-infected mice were abolished by exogenous 16,16-dimethyl PGE2. In conclusion, PGE2derived from either COX-1 or -2 is involved in regulation of gastric mucosal inflammation and contributes to maintenance of mucosal integrity during H. pylori infection via inhibition of TNF-α expression.
Mechanisms of diarrhea in the interleukin-2-deficient mouse model of colonic inflammation
Authors: Barmeyer C. , Harren M. , Schmitz H. , Heinzel-Pleines U. , Mankertz J. , Seidler U. , Horak I. , Wiedenmann B. , Fromm M. , Schulzke J. D.
Published: January 2004

Colitis in interleukin-2-deficient (IL-2-/-) mice resembles ulcerative colitis in humans. We studied epithelial transport and barrier function in IL-2-/-mice and used this model to characterize mechanisms of diarrhea during intestinal inflammation.22Na+and36Cl-fluxes were measured in proximal colon. Net Na+flux was reduced from 4.0 ± 0.5 to 0.8 ± 0.5 μmol·h-1·cm-2, which was paralleled by diminished mRNA and protein expression of the Na+/H+exchanger NHE3. Net Cl-flux was also decreased from 2.2 ± 1.6 to -2.7 ± 0.6 μmol·h-1·cm-2, indicating impaired Na+-Cl-absorption. In distal colon, aldosterone-induced electrogenic Na+absorption was 6.1 ± 0.9 μmol·h-1·cm-2in controls and was abolished in IL-2-/-mice. Concomitantly, mRNA expression of β- and γ-subunits of the epithelial sodium channel (ENaC) was reduced. Epithelial barrier was studied in proximal colon by impedance technique and mannitol fluxes. In contrast to ulcerative colitis, epithelial resistance was increased and mannitol fluxes were decreased in IL-2-/-mice. This was in accord with the findings of reduced ion transport as well as increased expression of tight junction proteins occludin and claudin-1, -2, -3, and -5. In conclusion, the IL-2-/-mucosa exhibits impaired electroneutral Na+-Cl-absorption and electrogenic Na+transport due to reduced mRNA and protein expression of NHE3 and ENaC β- and γ-subunit mRNA. This represents a model of early intestinal inflammation with absorptive dysfunction due to impaired transport protein expression/function while epithelial barrier is still intact. Therefore, this model is ideal to study regulation of transporter expression independent of barrier defects.
Regulation of butyrate uptake in Caco-2 cells by phorbol 12-myristate 13-acetate
Authors: Alrefai W. A. , Tyagi S. , Gill R. , Saksena S. , Hadjiagapiou C. , Mansour F. , Ramaswamy K. , Dudeja P. K.
Published: January 2004

Butyrate and the other short-chain fatty acids (SCFAs) are the most abundant anions in the colonic lumen. Also, butyrate is the preferred energy source for colonocytes and has been shown to regulate colonic electrolyte and fluid absorption. Previous studies from our group have demonstrated that the [Formula: see text] anion exchange process is one of the major mechanisms of butyrate transport across the purified human colonic apical membrane vesicles and the apical membrane of human colonic adenocarcinoma cell line Caco-2 and have suggested that it is mainly mediated via monocarboxylate transporter-1 (MCT-1) isoform. However, little is known regarding the regulation of SCFA transport by various hormones and signal transduction pathways. Therefore, the present studies were undertaken to examine whether hydrocortisone and phorbol 12-myristate 13-acetate (PMA) are involved in a possible regulation of the butyrate/anion exchange process in Caco-2 cells. The butyrate/anion exchange process was assessed by measuring a pH-driven [14C]butyrate uptake in Caco-2 cells. Our results demonstrated that 24-h incubation with PMA (1 μM) significantly increased [14C]butyrate uptake compared with incubation with 4αPMA (inactive form). In contrast, incubation with hydrocortisone had no significant effect on butyrate uptake in Caco-2 cells compared with vehicle (ethanol) alone. Induction of butyrate uptake by PMA appeared to be via an increase in the maximum velocity ( Vmax) of the transport process with no significant changes in the Kmof the transporter for butyrate. Parallel to the increase in the Vmaxof [14C]butyrate uptake, the MCT-1 protein level was also increased in response to PMA incubation. Our studies demonstrated that the butyrate/anion exchange was increased in response to PMA treatment along with the induction in the level of MCT-1 expression in Caco-2 cells.
Cl-/HCO3-exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca2+]iincrease in salivary acinar cells
Authors: Nguyen Ha-Van , Stuart-Tilley Alan , Alper Seth L. , Melvin James E.
Published: January 2004

Large volumes of saliva are generated by transepithelial Cl-movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl-uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na+independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an ∼170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive [Formula: see text] exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the [Formula: see text] exchanger activity in acinar cells from both glands. Intracellular Ca2+chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca2+concentration with the Ca2+-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating [Formula: see text] exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca2+-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.
PACAP and gastrin regulate the histidine decarboxylase promoter via distinct mechanisms
Authors: McLaughlin John T. , Ai Wandong , Sinclair Natalie F. , Colucci Rocchina , Raychowdhury Raktima , Koh Theodore J. , Wang Timothy C.
Published: January 2004

The enterochromaffin-like (ECL) cell controls gastric acid secretion via histamine, generated by l-histidine decarboxylase (HDC). HDC expression is regulated by gastrin. However, gastrin is not alone in controlling ECL cell function. For example, the neural peptide pituitary adenylate cyclase-activating polypeptide (PACAP) also increases ECL cell proliferation. To investigate a potential role of PACAP in regulating HDC expression, we generated a series of HDC promoter-luciferase reporter constructs and transiently transfected them into PC12 cells (stably expressing the gastrin-CCK-2 receptor). We found that PACAP regulates HDC promoter activity. This is temporally biphasic, involving both adenyl cyclase and phospholipase C-dependent pathways. Deletional analysis, block mutation, and EMSA demonstrated a PACAP-response element at -177 to -170, wholly necessary for the effects of PACAP and discrete from known gastrin-responsive elements. Discrete neural and endocrine pathways regulate ECL cells through different patterns of postreceptor signaling and promoter activation, which may be appropriate to their functions in vivo.
Reflex control of intestinal gas dynamics and tolerance in humans
Authors: Harder Hermann , Serra Jordi , Azpiroz Fernando , Malagelada Juan-R.
Published: January 2004

Intestinal transit of gas is normally adapted to the luminal gas load, but in some patients impaired transit may lead to gas retention and symptoms. We hypothesized that intestinal gas transit is regulated by reflex mechanisms released by segmental distension at various gut levels. In 24 healthy subjects, we measured gas evacuation and perception of jejunal gas infusion (12 ml/min) during simultaneous infusion of duodenal lipids mimicking the postprandial caloric load (Intralipid, 1 kcal/min). We evaluated the effects of proximal (duodenal) distension ( n = 8), distal (rectal) distension ( n = 8), and sham distension, as control ( n = 8). Duodenal lipid infusion produced gas retention (366 ± 106 ml) with low abdominal perception (1.5 ± 0.8 score). Distension of either the duodenum or rectum during lipid infusion expedited gas transit and prevented retention (-120 ± 164 and -124 ± 162 ml retention, respectively; P < 0.05 vs. control). However, the tolerance to the intestinal gas load differed markedly, depending on the site of distension; perception remained low during rectal distension (2.6 ± 0.7 score; not significant vs. control) but increased during duodenal distension (4.4 ± 0.7 score; P < 0.05 vs. control). We conclude that focal gut distension, either at proximal or distal sites, accelerates gas transit, but the symptomatic response depends on the site of stimulation.
Platelet-activating factor-induced apoptosis is blocked by Bcl-2 in rat intestinal epithelial cells
Authors: Lu Jing , Caplan Michael S. , Saraf Anita P. , Li Dan , Adler Luba , Liu Xuesong , Jilling Tamas
Published: January 2004

Plateletactivating factor (PAF) is a key mediator in pathogenesis of inflammatory bowel diseases (IBDs) but mechanisms of PAF-induced mucosal injury are poorly understood. To determine whether apoptosis and the Bcl-2-family of apoptosis regulatory gene products play a role in PAF-induced mucosal injury, we stably and conditionally overexpressed bcl-2 in rat small intestinal epithelial cells-6 under the control of a lactose-inducible promoter. Western blot analysis and immuno-histochemistry were used to verify inducible Bcl-2 and to analyze Bcl-2 and a proapoptotic member of the Bcl-2 family, Bax, subcellular distribution. DNA fragmentation was quantified by ELISA, caspase activity was measured by using fluorogenic peptide substrates, and mitochondrial membrane potential was assayed by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and fluorescence digital imaging. Bcl-2 expression was highly inducible by lactose analog isopropyl-β-d-thiogalactoside (IPTG) and was localized predominantly to mitochondria. In the absence of bcl-2 overexpression and after treatment with PAF, Bax translocated to mitochondria, and mitochondrial membrane potential collapsed within 1 h, followed by caspase-3 activation, which peaked at 6 h with an ensuing DNA fragmentation maximizing at 18 h. After IPTG-induction of bcl-2 expression, PAF failed to induce DNA fragmentation, caspase-3 activation, Bax translocation, or a collapse of mitochondrial membrane potential. These data are the first to show that PAF can activate apoptotic machinery in enterocytes via a mechanism involving Bax translocation and collapse of mitochondrial membrane potential and that both of these events are under control by bcl-2 expression levels. A better understanding of the role of PAF and Bcl-2 family of apoptosis regulators in epithelial cell death might aid design of better therapeutic or preventive strategies for IBDs.