bluuebunny/crossref_metadata_2025 huggingface dataset:
Amiloride-sensitive epithelial Na+ channel currents in surface cells of rat rectal colon
Authors: Inagaki A. , Yamaguchi S. , Ishikawa T.
Published: January 2004

Surface cells of the mammalian distal colon are shown to molecularly express the amiloride-sensitive epithelial Na+ channel composed of three homologous subunits (α-, β-, and γ-ENaC). However, because basic electrophysiological properties of amiloride-sensitive Na+ channels expressed in these cells are largely unknown at the cellular level, functional evidence for the involvement of the subunits in the native channels is incomplete. Using electrophysiological techniques, we have now characterized functional properties of native ENaC in surface cells of rectal colon (RC) of rats fed a normal Na+ diet. Ussing chamber experiments showed that apical amiloride inhibited a basal short-circuit current in mucosal preparation of RC with an apparent half-inhibition constant ( Ki) value of 0.20 μM. RT-PCR analysis confirmed the presence of transcripts of α-, β-, and γ-rENaC in rectal mucosa. Whole cell patch-clamp experiments in surface cells of intact crypts acutely isolated from rectal mucosa identified an inward cationic current, which was inhibited by amiloride with a Ki value of 0.12 μM at a membrane potential of –64 mV, the inhibition being weakly voltage dependent. Conductance ratios of the currents were Li+ (1.8) > Na+ (1) >> K+ (≈0), respectively. Amiloride-sensitive current amplitude was almost the same at 15 or 150 mM extracellular Na+, suggesting a high Na+ affinity for current activation. These results are consistent with the hypothesis that a heterooligomer composed of α-, β-, and γ-ENaC may be the molecular basis of the native channels, which are responsible for amiloride-sensitive electrogenic Na+ absorption in rat rectal colon.
System-specific O2 sensitivity of the tandem pore domain K+ channel TASK-1
Authors: Johnson Rosalyn P. , O'Kelly Ita M. , Fearon Ian M.
Published: January 2004

Hypoxic inhibition of TASK-1, a tandem pore domain background K+ channel, provides a critical link between reduced O2 levels and physiological responses in various cell types. Here, we examined the expression and O2 sensitivity of TASK-1 in immortalized adrenomedullary chromaffin (MAH) cells. In physiological (asymmetrical) K+ solutions, 3 μM anandamide or 300 μM Zn2+ inhibited a strongly pH-sensitive current. Under symmetrical K+ conditions, the anandamide- and Zn2+-sensitive K+ currents were voltage independent. These data demonstrate the functional expression of TASK-1, and cellular expression of this channel was confirmed by RT-PCR and Western blotting. At concentrations that selectively inhibit TASK-1, anandamide and Zn2+ were without effect on the magnitude of the O2-sensitive current or the hypoxic depolarization. Thus TASK-1 does not contribute to O2 sensing in MAH cells, demonstrating the failure of a known O2-sensitive K+ channel to respond to hypoxia in an O2-sensing cell. These data demonstrate that, ultimately, the sensitivity of a particular K+ channel to hypoxia is determined by the cell, and we propose that this is achieved by coupling distinct hypoxia signaling systems to individual channels. Importantly, these data also reiterate the indirect O2 sensitivity of TASK-1, which appears to require the presence of an intracellular mediator.
Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts
Authors: Emmert Daniel A. , Fee Judy A. , Goeckeler Zoe M. , Grojean Jeremy M. , Wakatsuki Tetsuro , Elson Elliot L. , Herring B. Paul , Gallagher Patricia J. , Wysolmerski Robert B.
Published: January 2004

Thus far, determining the relative contribution of Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and Ca2+-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca2+, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 ± 0.02 mol PO4/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 ± 0.05 and 0.82 ± 0.05 mol PO4/mol RLC, respectively, within 2.5 min. Ca2+ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells.
Metabolic organization in vascular smooth muscle: distribution and localization of caveolin-1 and phosphofructokinase
Authors: Vallejo Johana , Hardin Christopher D.
Published: January 2004

We have shown that a compartmentation of glycolysis and gluconeogenesis exists in vascular smooth muscle (VSM) and that an intact plasma membrane is essential for compartmentation. Previously, we observed that disruption of the caveolae inhibited glycolysis but stimulated gluconeogenesis, suggesting a link between caveolae and glycolysis. We hypothesized that glycolytic enzymes specifically localize to caveolae. We used confocal microscopy to determine the localization of caveolin-1 (CAV-1) and phosphofructokinase (PFK) in freshly isolated VSM cells and cultured A7r5 cells. Freshly isolated cells exhibited a peripheral (membrane) localization of CAV-1 with 85.3% overlap with PFK. However, only 59.9% of PFK was localized with CAV-1, indicating a wider distribution of PFK than CAV-1. A7r5 cells exhibited compartmentation of glycolysis and gluconeogenesis and displayed two apparent phenotypes distinguishable by shape (spindle and ovoid shaped). In both phenotypes, CAV-1 fluorescence overlapped with PFK fluorescence (83.1 and 81.5%, respectively). However, the overlap of PFK with CAV-1 was lower in the ovoid-shaped (35.9%) than the spindle-shaped cells (53.7%). There was also a progressive shift in pattern of colocalization from primarily the membrane in spindle-shaped cells (both freshly isolated and cultured cells) to primarily the cytoplasm in ovoid-shaped cells. Overall, cellular colocalization of PFK with CAV-1 was significant in all cell types (0.68 ≥ R2 ≤ 0.77). Coimmunoprecipitation of PFK with CAV-1 further validated the possible interaction between the proteins. We conclude that a similar distribution of one pool of PFK with CAV-1 contributes to the compartmentation of glycolysis from gluconeogenesis.
Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line
Authors: Bertrand C. A. , Danahay H. , Poll C. T. , Laboisse C. , Hopfer U. , Bridges R. J.
Published: January 2004

ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 ± 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 ± 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium ([Formula: see text]) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in [Formula: see text] was unaffected, but the initial response and overall time to [Formula: see text] peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.
Chloride-dependent calcium transients induced by angiotensin II in vascular smooth muscle cells
Authors: Ma Yunn-Hwa , Wei Hsiao-Wen , Su Kwan-Hwa , Ives Harlan E. , Morris R. Curtis
Published: January 2004

Cl– is essential for the vasoconstrictive response to angiotensin II (ANG II). In vascular smooth muscle cells (VSMC), we determined whether ANG II-induced transient increase in intracellular Ca2+ concentration ([Ca2+]i) is Cl– dependent. After incubating the cells at different extracellular Cl– concentration ([Cl–]e) for 40 min, the ANG II-induced Ca2+ transients at 120 meq/l Cl– were more than twice those at either 80 or 20 meq/l Cl–. Replacing Cl– with bicarbonate or gluconate yielded similar results. In addition, after removal of extracellular Ca2+, ANG II-induced as well as platelet-derived growth factor-induced Ca2+ release exhibited Cl– dependency. The difference of Ca2+ release with high vs. low [Cl–]e was not affected by acutely altering [Cl–]e 1 min before administration of ANG II when [Cl–]i was yet to be equilibrated with [Cl–]e. Pretreatment of a Cl– channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid, increased ANG II-induced Ca2+ release and entry at 20 meq/l Cl– but did not alter those at 120 meq/l Cl–. However, after equilibration, a reduced [Cl–]e did not affect thapsigargin-induced Ca2+ release, suggesting that Cl– may not affect the size of intracellular Ca2+ stores. Nevertheless, at high [Cl–], the peak increase of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] induced by ANG II was approximately sixfold that at low [Cl–]. Thus the Cl–-dependent effects of ANG II on Ca2+ transients may be mediated, at least in part, by a Cl–-dependent Ins(1,4,5)P3 accumulation in VSMC.
Effects of acute bouts of running and swimming exercise on PGC-1α protein expression in rat epitrochlearis and soleus muscle
Authors: Terada Shin , Tabata Izumi
Published: January 2004

The purpose of this study was to elucidate the mechanisms underlying low-intensity exercise-induced peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) protein expression in rat skeletal muscles. Rats (5–6 wk old) swam without a load and ran on the treadmill at a speed of 13 m/min, respectively, in two 3-h sessions separated by 45 min of rest. PGC-1α content in epitrochlearis muscle (EPI) was increased by 75 and 95%, immediately and 6 h after swimming, respectively, with no increase in PGC-1α content in the soleus (SOL). After running, PGC-1α content in EPI was unchanged, whereas a 107% increase in PGC-1α content was observed in SOL 6 h after running. Furthermore, in EPI and SOL as well as other muscles (triceps, plantaris, red and white gastrocnemius), PGC-1α expression was enhanced concomitant with reduced glycogen postexercise, suggesting that expression of PGC-1α occurs in skeletal muscle recruited during exercise. PGC-1α content in EPI was increased after 18-h in vitro incubation with 0.5 mM 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and 4 mM caffeine. However, AICAR incubation did not affect PGC-1α content in the SOL, whereas caffeine incubation increased it. These results suggest that exercise-induced PGC-1α expression in skeletal muscle may be mediated by at least two exercise-induced signaling factors: AMPK activation and Ca2+elevation. The number of factors involved (both AMPK and Ca2+, or Ca2+only) in exercise-induced PGC-1α expression may differ among muscles.
Increased systemic and adipose tissue cytokines in patients with HIV-associated lipodystrophy
Authors: Johnson Julia A. , Albu Jeanine B. , Engelson Ellen S. , Fried Susan K. , Inada Yoritaro , Ionescu Gabriel , Kotler Donald P.
Published: January 2004

The lipodystrophy syndrome (adipose tissue redistribution and metabolic abnormalities) observed with highly active antiretroviral therapy (HAART) during human immunodeficiency virus (HIV) infection may be related to increased proinflammatory cytokine activity. We measured acute cytokine (TNF-α, IL-6, leptin), glycerol, and lactate secretion from abdominal subcutaneous adipose tissue (SAT), and systemic cytokine levels, in HIV-infected subjects with and without lipodystrophy (HIVL+ and HIVL–, respectively) and healthy non-HIV controls. Lipodystrophy was confirmed and characterized as adipose tissue redistribution in HIVL+ compared with HIVL– and controls, by dual-energy X-ray absorptiometry and by whole body MRI. TNF-α secretion from abdominal SAT and circulating levels of IL-6, soluble TNF receptors I and II, and insulin were elevated in HIVL+ relative to HIVL– and/or controls, particularly in HIVL+ undergoing HAART. In the HIV-infected group as a whole, IL-6 secretion from abdominal SAT and serum IL-6 were positively associated with visceral fat and were negatively associated with the relative amount of lower limb adipose tissue ( P < 0.01). Decreased leptin and increased lactate secretion from abdominal SAT were specifically associated with HAART. In conclusion, increased cytokine secretion from adipose tissue and increased systemic proinflammatory cytokine activity may play a significant role in the adipose tissue remodeling and/or the metabolic abnormalities associated with the HIV-lipodystrophy syndrome in patients undergoing HAART.
Expression profiling of the γ-subunit isoforms of AMP-activated protein kinase suggests a major role for γ3 in white skeletal muscle
Authors: Mahlapuu Margit , Johansson Carina , Lindgren Kerstin , Hjälm Göran , Barnes Brian R. , Krook Anna , Zierath Juleen R. , Andersson Leif , Marklund Stefan
Published: January 2004

Expression patterns of the three isoforms of the regulatory γ-subunit of AMP-activated protein kinase (AMPK) were determined in various tissues from adult humans, mice, and rats, as well as in human primary muscle cells. Real-time PCR-based quantification of mRNA showed similar expression patterns in the three species and a good correlation with protein expression in mice and rats. The γ3-isoform appeared highly specific to skeletal muscle, whereas γ1 and γ2 showed broad tissue distributions. Moreover, the proportion of white, type IIb fibers in the mouse and rat muscle samples, as indicated by real-time PCR quantification of Atp1b2 mRNA, showed a strong positive correlation with the expression of γ3. In samples of white skeletal muscle, γ3 clearly appeared to be the most abundant γ-isoform. Differentiation of human primary muscle cells from myoblasts into multinucleated myotubes was accompanied by upregulation of γ3 mRNA expression, whereas levels of γ1 and γ2 remained largely unchanged. However, even in these cultured myotubes, γ2 was the most highly expressed isoform, indicating a considerable difference compared with adult skeletal muscle. Immunoblot analysis of mouse gastrocnemius and quadriceps muscle extracts precipitated with a γ3-specific antibody showed that γ3 was exclusively associated with the α2- and β2-subunit isoforms. The observation that the AMPKγ3 isoform is expressed primarily in white skeletal muscle, in which it is the predominant γ-isoform, strongly suggests that γ3 has a key role in this tissue.
NO plays a role in LPS-induced decreases in circulating IGF-I and IGFBP-3 and their gene expression in the liver
Authors: Priego Teresa , Ibáñez de Cáceres Inmaculada , Martín Ana Isabel , Villanúa M. Ángeles , López-Calderón Asunción
Published: January 2004

In this study, we administered aminoguanidine, a relatively selective inducible nitric oxide synthase (iNOS) inhibitor, to study the role of nitric oxide (NO) in LPS-induced decrease in IGF-I and IGFBP-3. Adult male Wistar rats were injected intraperitoneally with LPS (100 μg/kg), aminoguanidine (100 mg/kg), LPS plus aminoguanidine, or saline. Rats were injected at 1730 and 0830 the next day and killed 4 h after the last injection. LPS administration induced an increase in serum concentrations of nitrite/nitrate ( P < 0.01) and a decrease in serum concentrations of growth hormone (GH; P < 0.05) and IGF-I ( P < 0.01) as well as in liver IGF-I mRNA levels ( P < 0.05). The LPS-induced decrease in serum concentrations of IGF-I and liver IGF-I gene expression seems to be secondary to iNOS activation, since aminoguanidine administration prevented the effect of LPS on circulating IGF-I and its gene expression in the liver. In contrast, LPS-induced decrease in serum GH was not prevented by aminoguanidine administration. LPS injection decreased IGFBP-3 circulating levels ( P < 0.05) and its hepatic gene expression ( P < 0.01), but endotoxin did not modify the serum IGFBP-3 proteolysis rate. Aminoguanidine administration blocked the inhibitory effect of LPS on both IGFBP-3 serum levels and its hepatic mRNA levels. When aminoguanidine was administered alone, IGFBP-3 serum levels were increased ( P < 0.05), whereas its hepatic mRNA levels were decreased. This contrast can be explained by the decrease ( P < 0.05) in serum proteolysis of this binding protein caused by aminoguanidine. These data suggest that iNOS plays an important role in LPS-induced decrease in circulating IGF-I and IGFBP-3 by reducing IGF-I and IGFBP-3 gene expression in the liver.
Antenatal glucocorticoids reset the level of baseline and hypoxemia-induced pituitary-adrenal activity in the sheep fetus during late gestation
Authors: Fletcher Andrew J. W. , Ma Xiao Hong , Wu Wen X. , Nathanielsz Peter W. , McGarrigle Hugh H. G. , Fowden Abigail L. , Giussani Dino A.
Published: January 2004

This study examined the effects of dexamethasone treatment on basal hypothalamo-pituitary-adrenal (HPA) axis function and HPA responses to subsequent acute hypoxemia in the ovine fetus during late gestation. Between 117 and 120 days (term: ∼145 days), 12 fetal sheep and their mothers were catheterized under halothane anesthesia. From 124 days, 6 fetuses were continuously infused intravenously with dexamethasone (1.80 ± 0.15 μg·kg–1·h–1 in 0.9% saline at 0.5 ml/h) for 48 h, while the remaining 6 fetuses received saline at the same rate. Two days after infusion, when dexamethasone had cleared from the fetal circulation, acute hypoxemia was induced in both groups for 1 h by reducing the maternal fraction of inspired O2. Fetal dexamethasone treatment transiently lowered fetal basal plasma cortisol, but not ACTH, concentrations. However, 2 days after treatment, fetal basal plasma cortisol concentration was elevated without changes in basal ACTH concentration. Despite elevated basal plasma cortisol concentration, the ACTH response to acute hypoxemia was enhanced, and the increment in plasma cortisol levels was maintained, in dexamethasone-treated fetuses. Correlation of fetal plasma ACTH and cortisol concentrations indicated enhanced cortisol output without a change in adrenocortical sensitivity. The enhancements in basal cortisol concentration and the HPA axis responses to acute hypoxemia after dexamethasone treatment were associated with reductions in pituitary and adrenal glucocorticoid receptor mRNA contents, which persisted at 3–4 days after the end of treatment. These data show that prenatal glucocorticoids alter the basal set point of the HPA axis and enhance HPA axis responses to acute stress in the ovine fetus during late gestation.
High-fat diet elevates resting intramuscular triglyceride concentration and whole body lipolysis during exercise
Authors: Zderic Theodore W. , Davidson Christopher J. , Schenk Simon , Byerley Lauri O. , Coyle Edward F.
Published: January 2004

This study determined the role of intramuscular triglyceride (IMTG) and adipose lipolysis in the elevated fat oxidation during exercise caused by a high-fat diet. In four separate trials, six endurance-trained cyclists exercised at 50% peak O2consumption for 1 h after a two-day control diet (22% fat, CON) or an isocaloric high-fat diet (60% fat, HF) with or without the ingestion of acipimox, an adipose lipolysis inhibitor, before exercise. During exercise, HF elevated fat oxidation by 72% and whole body lipolysis [i.e., the appearance rate of glycerol in plasma (Raglycerol)] by 79% compared with CON ( P < 0.05), and this was associated with a 36% increase ( P < 0.05) in preexercise IMTG concentration. Although acipimox lowered plasma free fatty acid (FFA) availability, HF still increased fat oxidation and Raglycerol to the same magnitude above control as the increase caused by HF without acipimox (i.e., both increased fat oxidation 13–14 μmol·kg–1·min–1). In conclusion, the marked increase in fat oxidation after a HF diet is associated with elevated IMTG concentration and whole body lipolysis and does not require increased adipose tissue lipolysis and plasma FFA concentration during exercise. This suggests that altered substrate storage in skeletal muscle is responsible for increased fat oxidation during exercise after 2 days of an HF diet.
Autocrine regulation of glucose metabolism in pancreatic islets
Authors: Borelli María I. , Francini Flavio , Gagliardino Juan José
Published: January 2004

We evaluated the possible autocrine modulatory effect of insulin on glucose metabolism and glucose-induced insulin secretion in islets isolated from normal hamsters. We measured14CO2and3H2O production from d-[U-14C]glucose and d-[5-3H]glucose, respectively, in islets incubated with 0.6, 3.3, 8.3, and 16.7 mM glucose alone or with 5 or 15 mU/ml insulin, anti-insulin guinea pig serum (1:500), 25 μM nifedipine, or 150 nM wortmannin. Insulin release was measured (radioimmunoassay) in islets incubated with 3.3 or 16.7 mM glucose with or without 75, 150, and 300 nM wortmannin. Insulin significantly enhanced14CO2and3H2O production with 3.3 mM glucose but not with 0.6, 8.3, or 16.7 mM glucose. Addition of anti-insulin serum to the medium with 8.3 and 16.7 mM glucose decreased14CO2and3H2O production significantly. A similar decrease was obtained in islets incubated with 8.3 and 16.7 mM glucose and wortmannin or nifedipine. This latter effect was reversed by adding 15 mU/ml insulin to the medium. Glucose metabolism was almost abolished when islets were incubated in a Ca2+-deprived medium, but this effect was not reversed by insulin. No changes were found in14CO2and3H2O production by islets incubated with 3.3 mM glucose and anti-insulin serum, wortmannin, or nifedipine in the media. Addition of wortmannin significantly decreased insulin release induced by 16.7 mM glucose in a dose-dependent manner. Our results suggest that insulin exerts a physiological autocrine stimulatory effect on glucose metabolism in intact islets as well as on glucose-induced insulin release. Such an effect, however, depends on the glucose concentration in the incubation medium.
Glucose ingestion blunts hormone-sensitive lipase activity in contracting human skeletal muscle
Authors: Watt Matthew J. , Krustrup Peter , Secher Niels H. , Saltin Bengt , Pedersen Bente K. , Febbraio Mark A.
Published: January 2004

To examine the effect of attenuated epinephrine and elevated insulin on intramuscular hormone sensitivity lipase activity (HSLa) during exercise, seven men performed 120 min of semirecumbent cycling (60% peak pulmonary oxygen uptake) on two occasions while ingesting either 250 ml of a 6.4% carbohydrate (GLU) or sweet placebo (CON) beverage at the onset of, and at 15 min intervals throughout, exercise. Muscle biopsies obtained before and immediately after exercise were analyzed for HSLa. Blood samples were simultaneously obtained from a brachial artery and a femoral vein before and during exercise, and leg blood flow was measured by thermodilution in the femoral vein. Net leg glycerol and lactate release and net leg glucose and free fatty acid (FFA) uptake were calculated from these measures. Insulin and epinephrine were also measured in arterial blood before and throughout exercise. During GLU, insulin was elevated (120 min: CON, 11.4 ± 2.4, GLU, 35.3 ± 6.9 pM, P < 0.05) and epinephrine suppressed (120 min: CON, 6.1 ± 2.5, GLU, 2.1 ± 0.9 nM; P < 0.05) compared with CON. Carbohydrate feeding also resulted in suppressed ( P < 0.05) HSLa relative to CON (120 min: CON, 1.71 ± 0.18, GLU, 1.27 ± 0.16 mmol·min-1·kg dry mass-1). There were no differences in leg lactate or glycerol release when trials were compared, but leg FFA uptake was lower (120 min: CON, 0.29 ± 0.06, GLU, 0.82 ± 0.09 mmol/min) and leg glucose uptake higher (120 min: CON, 3.16 ± 0.59, GLU, 1.37 ± 0.37 mmol/min) in GLU compared with CON. These results demonstrate that circulating insulin and epinephrine play a role in HSLa in contracting skeletal muscle.
Streptozotocin-induced diabetes impairs Mg2+homeostasis and uptake in rat liver cells
Authors: Fagan Theresa E. , Cefaratti Christie , Romani Andrea
Published: January 2004

Male Sprague-Dawley rats rendered diabetic by streptozotocin injection presented 10 and 20% decreases in total hepatic Mg2+content at 4 and 8 wk, respectively, following diabetes onset. This decrease was associated with a parallel decrease in K+and ATP content and an increase in Na+level. In diabetic liver cells, the Mg2+extrusion elicited by α1-adrenoceptor stimulation was markedly reduced compared with nondiabetic livers, whereas that induced by β-adrenoceptor stimulation was unaffected. In addition, diabetic hepatocytes did not accumulate Mg2+following stimulation of protein kinase C pathway by vasopressin, diacylglycerol analogs, or phorbol 12-myristate 13-acetate derivates despite the reduced basal content in cellular Mg2+. Experiments performed in purified plasma membrane from diabetic livers located the defect at the level of the bidirectional Na+/Mg2+exchanger operating in the basolateral domain of the hepatocyte cell membrane, which could extrude but not accumulate Mg2+in exchange for Na+. The impairment of Mg2+uptake mechanism, in addition to the decrease in cellular ATP level, can contribute to explaining the decrease in liver Mg2+content observed under diabetic conditions.
Comparison of intraduodenal and intravenous glucose metabolism under clamp conditions in humans
Authors: Féry F. , Tappy L. , Devière J. , Balasse E. O.
Published: January 2004

To determine whether the uptake and metabolic partition of glucose are influenced by its delivery route, 12 normal volunteers underwent two 3-h euglycemic (∼93 mg/dl) hyperinsulinemic (∼43 mU/l) clamps at a 3- to 5-wk interval, one with intravenous (IV) and the other with intraduodenal (ID) glucose labeled with [3-3H]- and [U-14C]glucose. Systemic glucose was traced with [6,6-2H2]glucose in eight subjects. During the last hour of the clamps, the average glucose infusion rate (5.85 ± 0.37 vs. 5.43 ± 0.43 mg·kg–1·min–1; P = 0.02) and exogenous glucose uptake (5.66 ± 0.37 vs. 5.26 ± 0.41 mg·kg–1·min–1; P = 0.04) were borderline higher in the ID than in the IV studies. The increased uptake was entirely accounted for by increased glycolysis (3H2O production), which was attributed to the stimulation of gut metabolism by the absorptive process. No difference was observed in glucose storage whether it was calculated as glucose uptake minus glycolysis (ID vs. IV: 2.44 ± 0.28 vs. 2.40 ± 0.31 mg·kg–1·min–1) or as glucose uptake minus net glucose oxidation (2.86 ± 0.33 vs. 2.81 ± 0.35 mg·kg–1·min–1). Because peripheral tissues were exposed to identical glucose, insulin, and free fatty acid levels under the two experimental conditions, we assumed that their glucose uptake and storage were similar during the two tests. We therefore suggest that hepatic glycogen storage (estimated as whole body minus peripheral storage) was also unaffected by the route of glucose delivery. On the other hand, in the ID tests, the glucose splanchnic extraction ratio calculated by the dual-isotope technique averaged 4.9 ± 2.3%, which is close to the figures published for IV glucose. Despite the limitations related to whole body measurements, these two sets of data do not support the idea that enteral glucose stimulates hepatic uptake more efficiently than IV glucose.
Altered expression of nuclear hormone receptors and coactivators in mouse heart during the acute-phase response
Authors: Feingold Kenneth , Kim Min Sun , Shigenaga Judy , Moser Art , Grunfeld Carl
Published: January 2004

Severe sepsis results in the decreased uptake and oxidation of fatty acids in the heart and cardiac failure. Some of the key proteins required for fatty acid uptake and oxidation in the heart have been shown to be downregulated after endotoxin (LPS) administration. The nuclear hormone receptors, peroxisome proliferator-activated receptor (PPAR) and thyroid receptor (TR), which heterodimerize with the retinoid X receptor (RXR), are important regulators of fatty acid metabolism and decrease in the liver after LPS administration. In the present study, we demonstrate that LPS treatment produces a rapid and marked decrease in the mRNA levels of all three RXR isoforms, PPARα and PPARδ, and TRα and TRβ in the heart. Moreover, LPS administration also decreased the expression of the coactivators CREB-binding protein (CBP)/p300, steroid receptor coactivator (SRC)-1, SRC-3, TR-associated protein (TRAP)220, and PPARγ coactivator (PGC)-1, all of which are required for the transcriptional activity of RXR-PPAR and RXR-TR. In addition, the mRNA levels of the target genes malic enzyme, Spot 14, sarcoplasmic reticulum Ca2+-ATPase, or SERCA2, the VLDL receptor, fatty acyl-CoA synthetase, fatty acid transporter/CD36, carnitine palmitoyltransferase Iβ, and lipoprotein lipase decrease in the heart after LPS treatment. The decrease in expression of RXRα, -β, and -γ, PPARα and -δ, and TRα and -β, and of the coactivators CBP/p300, SRC-1, SRC-3, TRAP220, and PGC-1 and the genes they regulate, induced by LPS in the heart, could account for the decreased expression of key proteins required for fatty acid oxidation and thereby play an important role in cardiac contractility. These alterations could contribute to the myocardial dysfunction that occurs during sepsis.
Prolactin, progesterone, and dexamethasone coordinately and adversely regulate glucokinase and cAMP/PDE cascades in MIN6 β-cells
Authors: Shao Jianhua , Qiao Liping , Friedman Jacob E.
Published: January 2004

Islet cells undergo major changes in structure and function to meet the demand for increased insulin secretion during pregnancy, but the nature of the hormonal interactions and signaling events is incompletely understood. Here, we used the glucose-responsive MIN6 β-cell line treated with prolactin (PRL), progesterone (PRG), and dexamethasone (DEX, a synthetic glucocorticoid), all elevated during late pregnancy, to study their effects on mechanisms of insulin secretion. DEX alone or combined with PRL and PRG inhibited insulin secretion in response to 16 mM glucose-stimulating concentrations. However, in the basal state (3 mM glucose), the insulin levels in response to DEX treatment were unchanged, and the three hormones together maintained higher insulin release. There were no changes of protein levels of GLUT2 or glucokinase (GK), but PRL or PRG treatment increased GK activity, whereas DEX had an inhibitory effect on GK activity. α-Ketoisocaproate (α-KIC)-stimulated insulin secretion was also reduced by DEX alone or combined with PRL and PRG, suggesting that DEX may inhibit distal steps in the insulin-exocytotic process. PRL treatment increased the concentration of intracellular cAMP in response to 16 mM glucose, suggesting a role for cAMP in potentiation of insulin secretion, whereas DEX alone or combined with PRL and PRG reduced cAMP levels by increasing phosphodiesterase (PDE) activity. These data provide evidence that PRL and to a lesser extent PRG, which increase in early pregnancy, enhance basal and glucose-stimulated insulin secretion in part by increasing GK activity and amplifying cAMP levels. Glucocorticoid, which increases throughout gestation, counteracts only glucose-stimulated insulin secretion under high glucose concentrations by dominantly inhibiting GK activity and increasing PDE activity to reduce cAMP levels. These adaptations in the β-cell may play an important role in maintaining the basal hyperinsulinemia of pregnancy while limiting the capacity of PRL and PRG to promote glucose-stimulated insulin secretion during late gestation.
Body cell mass: model development and validation at the cellular level of body composition
Authors: Wang ZiMian , St-Onge Marie-Pierre , Lecumberri Beatriz , Pi-Sunyer F. Xavier , Heshka Stanley , Wang Jack , Kotler Donald P. , Gallagher Dympna , Wielopolski Lucian , Pierson Richard N. , Heymsfield Steven B.
Published: January 2004

Existing models to estimate the metabolically active body cell mass (BCM) component in vivo remain incompletely developed. The classic Moore model is based on an assumed BCM potassium content of 120 mmol/kg. Our objectives were to develop an improved total body potassium (TBK)-independent BCM prediction model on the basis of an earlier model (Cohn SH, Vaswani AN, Yasumura S, Yuen K, and Ellis KJ. J Lab Clin Med 105: 305-311, 1985), to apply this improved model in subjects to explore the sex and age dependence of the TBK/BCM ratio, to develop a new TBK/BCM model on the basis of physiological associations between TBK and total body water (TBW) at the cellular level of body composition, and to fit this new model with available reference data. Subjects were 112 healthy adults who had the following components measured: TBW by 2H2O or 3H2O, extracellular water by NaBr, total body nitrogen by in vivo neutron activation, bone mineral by dual-energy X-ray absorptiometry, and TBK by whole body counting. Human reference data were collected from earlier published reports. The improved Cohn model-derived TBK/BCM ratio was (mean ± SD) 109.0 ± 10.9 mmol/kg and was not significantly related to sex and age. A simplified version of the new TBK-TBW model provided a TBK/BCM ratio almost identical (109.1 mmol/kg) to that derived by the improved Cohn model. The TBK-BCM prediction formula derived from the improved and new models [BCM (kg) = 1/109 × TBK (mmol); or BCM = 0.0092 × TBK] gives BCM estimates ∼11% higher than the classic Moore model (BCM = 0.0083 × TBK) formulated on rough tissue composition estimates. The present analyses provide a physiologically based, improved, and validated TBK-BCM prediction formula that should prove useful in body composition and metabolism research.
Uncoupling protein-2 polymorphisms in type 2 diabetes, obesity, and insulin secretion
Authors: Wang Hua , Chu Winston S. , Lu Tong , Hasstedt Sandra J. , Kern Philip A. , Elbein Steven C.
Published: January 2004

The onset of type 2 diabetes (T2DM) is preceded by obesity, insulin resistance, and impaired β-cell function. Uncoupling protein-2 (UCP2) is a widely expressed inner mitochondrial membrane protein. Common polymorphisms of the UCP2 gene have been implicated in diabetes, in obesity, and with changes in UCP2 mRNA levels. We tested the hypothesis that common UCP2 variants influence T2DM susceptibility in four parallel studies of separate populations. We typed the -866 promoter (G/A) variant, a nonsynonymous (Ala55Val or A55V) single-nucleotide polymorphism in exon 4, and a 45-nt insertion in the 3′-untranslated (3′UTR) region. Study populations included a case-control population study, a family-based association study, and a metabolic study of individuals who had been characterized for insulin sensitivity and secretion. To evaluate UCP2 mRNA levels, we examined a fourth population of subjects, who had undergone subcutaneous fat biopsy. All three variants showed a trend to an association with T2DM ( P = 0.05 to 0.07) in the population but not the family-based association study. The 3′ insertion/deletion (3′UTR I/D) variant was associated with body mass index (BMI, P = 0.035) among nondiabetic family members. Haplotype combinations were significantly associated with BMI ( P = 0.028), triglyceride levels ( P = 0.026), and fasting insulin ( P = 0.029); highest values for the three traits were observed in individuals with the heterozygous combination GVI/AVD. In the metabolic study, all three variants were associated with an index of β-cell compensation for insulin sensitivity (disposition index), particularly in interaction with family membership ( P < 0.000001). Individuals homozygous for the -866 A allele had decreased adipose mRNA levels relative to GG homozygous individuals ( P = 0.009), but the 3′UTR I/D variant had no impact on mRNA levels. We confirm modest effects of UCP2 variants on BMI and T2DM and show significant effects on insulin secretion in interaction with family-specific factors. However, the associated allele and the effects on gene expression are opposite to those reported previously.
Human skeletal muscle PPARα expression correlates with fat metabolism gene expression but not BMI or insulin sensitivity
Authors: Zhang Junlong , Phillips D. I. W. , Wang Chunli , Byrne Christopher D.
Published: January 2004

Peroxisome proliferator-activated receptor-α (PPARα) is a key regulator of fatty acid oxidation in skeletal muscle, but few data exist from humans in vivo. To investigate whether insulin sensitivity in skeletal muscle and body mass index (BMI) were associated with skeletal muscle expression of PPARα and with important genes regulating lipid metabolism in humans in vivo, we undertook hyperinsulinemic-euglycemic clamps and measured PPARα mRNA levels and mRNA levels of lipid regulating PPARα response genes in skeletal muscle biopsies. mRNA levels were measured in 16 men, using a novel highly sensitive and specific medium throughput quantitative competitive PCR that allows reproducible measurement of multiple candidate mRNAs simultaneously. mRNA levels of PPARα were positively correlated with mRNA levels of CD36 ( r = 0.77, P = 0.001), lipoprotein lipase ( r = 0.54, P = 0.024), muscle-type carnitine palmitoyltransferase-I ( r = 0.54, P = 0.024), uncoupling protein-2 ( r = 0.63, P = 0.008), and uncoupling protein-3 ( r = 0.53, P = 0.026), but not with measures of insulin sensitivity, BMI, or GLUT4, which plays an important role in insulin-mediated glucose uptake. Thus our data suggest that in humans skeletal muscle PPARα expression and genes regulating lipid metabolism are tightly linked, but there was no association between both insulin sensitivity and BMI with PPARα expression in skeletal muscle.
Regulation of CPT I activity in intermyofibrillar and subsarcolemmal mitochondria from human and rat skeletal muscle
Authors: Bezaire Veronic , Heigenhauser George J. F. , Spriet Lawrence L.
Published: January 2004

Carnitine palmitoyltransferase I (CPT I) is considered the rate-limiting enzyme in the transfer of long-chain fatty acids (LCFA) into the mitochondria and is reversibly inhibited by malonyl-CoA (M-CoA) in vitro. In rat skeletal muscle, M-CoA levels decrease during exercise, releasing the inhibition of CPT I and increasing LCFA oxidation. However, in human skeletal muscle, M-CoA levels do not change during moderate-intensity exercise despite large increases in fat oxidation, suggesting that M-CoA is not the sole regulator of increased CPT I activity during exercise. In the present study, we measured CPT I activity in intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondria isolated from human vastus lateralis (VL), rat soleus (Sol), and red gastrocnemius (RG) muscles. We tested whether exercise-related levels (∼65% maximal O2uptake) of calcium and adenylate charge metabolites (free AMP, ADP, and Pi) could override the M-CoA-induced inhibition of CPT I activity and explain the increased CPT I flux during exercise. Protein content was ∼25-40% higher in IMF than in SS mitochondria in all muscles. Maximal CPT I activity was similar in IMF and SS mitochondria in all muscles (VL: 282 ± 46 vs. 280 ± 51; Sol: 390 ± 81 vs. 368 ± 82; RG: 252 ± 71 vs. 278 ± 44 nmol·min-1·mg protein-1). Sensitivity to M-CoA did not differ between IMF and SS mitochondria in all muscles (25-31% inhibition in VL, 52-70% in Sol and RG). Calcium and adenylate charge metabolites did not override the M-CoA-induced inhibition of CPT I activity in mitochondria isolated from VL, Sol, and RG muscles. Decreasing pH from 7.1 to 6.8 reduced CPT I activity by ∼34-40% in both VL mitochondrial fractions. In summary, this study reports no differences in CPT I activity or sensitivity to M-CoA between IMF and SS mitochondria isolated from human and rat skeletal muscles. Exercise-induced increases in calcium and adenylate charge metabolites do not appear responsible for upregulating CPT I activity in human or rat skeletal muscle during moderate aerobic exercise.
Branched-chain amino acid catabolism: unique segregation of pathway enzymes in organ systems and peripheral nerves
Authors: Sweatt Andrew J. , Wood Mac , Suryawan Agus , Wallin Reidar , Willingham Mark C. , Hutson Susan M.
Published: January 2004

We have examined the localization of the first two enzymes in the branched-chain amino acid (BCAA) catabolic pathway: the branched-chain aminotransferase (BCAT) isozymes (mitochondrial BCATm and cytosolic BCATc) and the branched-chain α-keto acid dehydrogenase (BCKD) enzyme complex. Antibodies specific for BCATm or BCATc were used to immunolocalize the respective isozymes in cryosections of rat tissues. BCATm was expressed in secretory epithelia throughout the digestive tract, with the most intense expression in the stomach. BCATm was also strongly expressed in secretory cells of the exocrine pancreas, uterus, and testis, as well as in the transporting epithelium of convoluted tubules in kidney. In muscle, BCATm was located in myofibrils. Liver, as predicted, was not immunoreactive for BCATm. Unexpectedly, BCATc was localized in elements of the autonomic innervation of the digestive tract, as well as in axons in the sciatic nerve. The distributions of BCATc and BCATm did not overlap. BCATm-expressing cells also expressed the second enzyme of the BCAA catabolic pathway, BCKD. In selected monkey and human tissues examined by immunoblot and/or immunohistochemistry, BCATm and BCATc were distributed in patterns very similar to those found in the rat. The results show that BCATm is in a position to regulate BCAA availability as protein precursors and anabolic signals in secretory portions of the digestive and other organ systems. The unique expression of BCATc in neurons of the peripheral nervous system, without coexpression of BCKD, raises new questions about the physiological function of this BCAT isozyme.
Effects of corticosterone on muscle mitochondria identifying different sensitivity to glucocorticoids in Lewis and Fischer rats
Authors: Duclos Martine , Gouarne Caroline , Martin Cyril , Rocher Christophe , Mormède Pierre , Letellier Thierry
Published: January 2004

Previous studies in rat have demonstrated decreased number of mitochondria and uncoupling of oxidative phosphorylation after administration of glucocorticoids but at supraphysiological doses and using synthetic glucocorticoids. To analyze the relationships between corticosterone levels (the natural glucocorticoid in rat) and muscle mitochondrial metabolism, Lewis and Fischer 344 rats were bilaterally adrenalectomized and implanted with different corticosterone pellets (0, 12, 50, 100, and 200 mg of corticosterone). Rats bearing a corticosterone pellet delivering corticosterone at concentrations in the range of chronic stress-induced levels presented a lower amount of functional muscle mitochondria with a decrease in cytochrome c oxidase and citrate synthase activities and a depletion of mitochondrial DNA. Moreover, a strain difference in tissue sensitivity to corticosterone was depicted both in end-organ sensitive to glucocorticoids (body, thymus, and adrenal weights) and in muscle mitochondrial metabolism (Lewis > Fischer). Interestingly, this strain difference was also observed in the absence of corticosterone, with a deleterious effect on muscle mitochondrial metabolism in Fischer rats, whereas no effects were observed in Lewis rats. We therefore postulate that corticosterone is necessary for muscle mitochondrial metabolism exerting its effects in Fischer rats with an inverted U curve, whereby too little (only Fischer) or too much (Fischer and Lewis) corticosterone is deleterious to muscle mitochondrial metabolism. In conclusion, we propose a general model of coordinate regulation of mitochondrial energetic metabolism by glucocorticoids.
Proton leak and hydrogen peroxide production in liver mitochondria from energy-restricted rats
Authors: Ramsey Jon J. , Hagopian Kevork , Kenny Teresa M. , Koomson Edward K. , Bevilacqua Lisa , Weindruch Richard , Harper Mary-Ellen
Published: January 2004

Energy restriction (ER), without malnutrition, is the only environmental intervention that consistently increases maximum life span in laboratory rodents. One theory proposes that a reduction in energy expenditure and reactive oxygen species production is the mechanism responsible for this action of ER. To further test this theory, proton leak, H2O2production, lipid peroxidation, and protein carbonyls were measured in mitochondria from FBNF1rats fed either a control or 40% ER diet (onset at 6 mo of age). Liver mitochondria were isolated at 7 and 12 mo of age. Liver weight decreased 25 and 36% at 1 and 6 mo of ER, respectively ( P < 0.05). ER resulted in an increase ( P < 0.05) in percent total polyunsaturates, n-6 polyunsaturates, and total unsaturates (6 mo only) in mitochondrial lipids. These changes, however, were not associated with significant alterations in mitochondrial function. State 4 respiration and membrane potential were not different ( P > 0.05) between groups at either assessment period. Similarly, proton leak kinetics were not different between control and ER animals. Top-down metabolic control analysis and its extension, elasticity analysis, were used at the 6-mo assessment and revealed no difference in control of the oxidative phosphorylation system between control and ER rats. H2O2production with either succinate or pyruvate/malate substrates was also not different ( P > 0.05) between groups at either time point. In conclusion, ER did not alter proton leak or H2O2production at this age or stage of restriction in liver.