bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessing menstrual cycles with urinary hormone assays
Authors: Santoro N. , Crawford S. L. , Allsworth J. E. , Gold E. B. , Greendale G. A. , Korenman S. , Lasley B. L. , McConnell D. , McGaffigan P. , Midgely R. , Schocken M. , Sowers M. , Weiss G.
Published: March 2015

The Study of Women's Health Across the Nation (SWAN) is a multiethnic cohort study of middle-aged women enrolled at seven US sites. A subset of 848 women completed a substudy in which their urinary gonadotropins and sex steroid metabolites were assessed during one complete menstrual cycle or up to 50 consecutive days. Urine was analyzed for LH, FSH, estrone conjugates (E1c), and pregnanediol glucuronide (Pdg). To prepare for serial analysis of this large, longitudinal database in a population of reproductively aging women, we examined the performance of algorithms designed to identify features of the normal menstrual cycle in midreproductive life. Algorithms were based on existing methods and were compared with a “gold standard” of ratings of trained observers on a subset of 396 cycles from the first collection of Daily Hormone Substudy samples. In evaluating luteal status, overall agreement between and within raters was high. Only 17 of the 396 cycles evaluated were considered indeterminate. Of the 328 cycles rated as containing evidence of luteal activity (ELA), 320 were considered ELA by use of a Pdg threshold detection algorithm. Of 51 cycles that were rated as no evidence of luteal activity, only 2 were identified by this algorithm as ELA. Evaluation of the day of the luteal transition with methods that detected a change in the ratio of E1c to Pdg provided 85–92% agreement for day of the luteal transition within 3 days of the raters. Adding further conditions to the algorithm increased agreement only slightly, by 1–8%. We conclude that reliable, robust, and relatively simple objective methods of evaluation of the probability and timing of ovulation can be used with urinary hormonal assays in early perimenopausal women.
Sumoylation of Pdx1 is associated with its nuclear localization and insulin gene activation
Authors: Kishi Akio , Nakamura Takaaki , Nishio Yoshihiko , Maegawa Hiroshi , Kashiwagi Atsunori
Published: March 2015

Pancreatic duodenal homeobox-1 (Pdx1) is a transcription factor, and its phosphorylation is thought to be essential for activation of insulin gene expression. This phosphorylation is related to a concomitant shift in molecular mass from 31 to 46 kDa. However, we found that Pdx1 was modified by SUMO-1 (small ubiquitin-related modifier 1) in β-TC-6 and COS-7 cells, which were transfected with Pdx1 cDNA. This modification contributed to the increase in molecular mass of Pdx1 from 31 to 46 kDa. Additionally, sumoylated Pdx1 localized in the nucleus. The reduction of SUMO-1 protein by use of RNA interference (SUMO-iRNAs) resulted in a significant decrease in Pdx1 protein in the nucleus. A 34-kDa form of Pdx1 was detected by the cells exposed to SUMO-iRNAs in the presence of lactacystin, a proteasome inhibitor. Furthermore, the reduced nuclear sumoylated Pdx1 content was associated with significant lower transcriptional activity of the insulin gene. These findings indicate that SUMO-1 modification is associated with both the localization and stability of Pdx1 as well as its effect on insulin gene activation.
Blood flow and muscle metabolism: a focus on insulin action
Authors: Clark Michael G. , Wallis Michelle G. , Barrett Eugene J. , Vincent Michelle A. , Richards Stephen M. , Clerk Lucy H. , Rattigan Stephen
Published: March 2015

The vascular system controls the delivery of nutrients and hormones to muscle, and a number of hormones may act to regulate muscle metabolism and contractile performance by modulating blood flow to and within muscle. This review examines evidence that insulin has major hemodynamic effects to influence muscle metabolism. Whole body, isolated hindlimb perfusion studies and experiments with cell cultures suggest that the hemodynamic effects of insulin emanate from the vasculature itself and involve nitric oxide-dependent vasodilation at large and small vessels with the purpose of increasing access for insulin and nutrients to the interstitium and muscle cells. Recently developed techniques for detecting changes in microvascular flow, specifically capillary recruitment in muscle, indicate this to be a key site for early insulin action at physiological levels in rats and humans. In the absence of increases in bulk flow to muscle, insulin may act to switch flow from nonnutritive to the nutritive route. In addition, there is accumulating evidence to suggest that insulin resistance of muscle in vivo in terms of impaired glucose uptake could be partly due to impaired insulin-mediated capillary recruitment. Exercise training improves insulin-mediated capillary recruitment and glucose uptake by muscle.
Gluconeogenesis in humans with induced hyperlactatemia during low-intensity exercise
Authors: Roef Mark J. , de Meer Kees , Kalhan Satish C. , Straver Helma , Berger Ruud , Reijngoud Dirk-Jan
Published: March 2015

We studied the role of lactate in gluconeogenesis (GNG) during exercise in untrained fasting humans. During the final hour of a 4-h cycle exercise at 33–34% maximal O2uptake, seven subjects received, in random order, either a sodium lactate infusion (60 μmol · kg−1· min−1) or an isomolar sodium bicarbonate infusion. The contribution of lactate to gluconeogenic glucose was quantified by measuring2H incorporation into glucose after body water was labeled with deuterium oxide, and glucose rate of appearance (Ra) was measured by [6,6-2H2]glucose dilution. Infusion of lactate increased lactate concentration to 4.4 ± 0.6 mM (mean ± SE). Exercise induced a decrease in blood glucose concentration from 5.0 ± 0.2 to 4.2 ± 0.3 mM ( P < 0.05); lactate infusion abolished this decrease (5.0 ± 0.3 mM; P < 0.001) and increased glucose Racompared with bicarbonate infusion ( P < 0.05). Lactate infusion increased both GNG from lactate (29 ± 4 to 46 ± 4% of glucose Ra, P < 0.001) and total GNG. We conclude that lactate infusion during low-intensity exercise in fasting humans 1) increased GNG from lactate and 2) increased glucose production, thus increasing the blood glucose concentration. These results indicate that GNG capacity is available in humans after an overnight fast and can be used to sustain blood glucose levels during low-intensity exercise when lactate, a known precursor of GNG, is available at elevated plasma levels.
Effect of endotoxin on expression of TNF receptors and transport of TNF-α at the blood-brain barrier of the rat
Authors: Osburg B. , Peiser C. , Dömling D. , Schomburg L. , Ko Y. T. , Voigt K. , Bickel U.
Published: March 2015

The transport mechanism mediating brain uptake of tumor necrosis factor (TNF)-α has been studied. When125I-labeled rat TNF-α was used in internal carotid artery perfusions in rats, the cytokine showed transcytosis through the blood-brain barrier in intact form (permeability-surface area product 0.34 ± 0.13 μl · min−1· g−1). Uptake was inhibited by low nanomolar concentrations of unlabeled rat TNF-α. Human TNF-α, which does not interact with the p80 TNF receptor in rodents, showed no brain uptake. mRNA expression of both p60 and p80 receptors could be demonstrated in native brain microvessel preparations. These transcripts increased to 149% (p60) and 127% (p80) of control 4 h after a systemic immune stimulation (2 mg/kg bacterial endotoxin ip). Lipopolysaccharide treatment did not alter the rate of brain uptake of TNF-α measured between 4 and 24 h later. In conclusion, a receptor-mediated mechanism is responsible for the transcytosis of TNF-α. Saturable transport, requiring the p80 receptor, occurs at concentrations encountered under pathophysiological conditions and therefore constitutes a relevant mechanism of communication between the immune system and the brain.
Impaired oxidative phosphorylation in hepatic mitochondria in growth-retarded rats
Authors: Peterside Iyalla E. , Selak Mary A. , Simmons Rebecca A.
Published: March 2015

Intrauterine growth retardation (IUGR) has been linked to the development of type 2 diabetes in adulthood. We have developed an IUGR model in the rat whereby the animals develop diabetes between 3 and 6 mo of age that is associated with insulin resistance. Alterations in hepatic glucose metabolism are known to contribute to the hyperglycemia of diabetes; however, the mechanisms underlying this phenomenon have not been fully explained. To address this issue, intact liver mitochondria were isolated from IUGR and control offspring at different ages to examine the nature and time course of possible defects in oxidative metabolism. Phospho enolpyruvate carboxykinase (PEPCK) expression was also measured in livers of IUGR and control offspring. Rates of ADP-stimulated (state 3) oxygen consumption were increased for succinate in the fetus and for α-ketoglutarate and glutamate at day 1, reflecting possible compensatory metabolic adaptations to acute hypoxia and acidosis in IUGR rats. By day 14, oxidation of glutamate and α-ketoglutarate had returned to normal, and by day 28, oxidation rates of pyruvate, glutamate, succinate, and α-ketoglutarate were significantly lower than those of controls. Rotenone-sensitive NADH-O2oxidoreductase activity was similar in control and IUGR mitochondria at all ages, showing that the defect responsible for decreased pyruvate, glutamate, and α-ketoglutarate oxidation in IUGR liver precedes the electron transport chain and involves pyruvate and α-ketoglutarate dehydrogenases. Increased levels of manganese superoxide dismutase suggest that an antioxidant response has been mounted, and hydroxynonenal (HNE) modification of pyruvate dehydrogenase E2-(catalytic) and E3-binding protein subunits suggests that HNE-induced inactivation of this key enzyme may play a role in the mechanism of injury. The level of PEPCK mRNA was increased 250% in day 28 IUGR liver, indicating altered gene expression of the gluconeogenic enzyme that precedes overt hyperglycemia. These results indicate that uteroplacental insufficiency impairs mitochondrial oxidative phosphorylation in the liver and that this derangement predisposes the IUGR rat to increased hepatic glucose production by suppressing pyruvate oxidation and increasing gluconeogenesis.
Expression of uncoupling protein 3 is upregulated in skeletal muscle during sepsis
Authors: Sun Xiaoyan , Wray Curtis , Tian Xintian , Hasselgren Per-Olof , Lu James
Published: March 2015

Uncoupling protein 3 (UCP3) is a member of the mitochondrial transporter superfamily that is expressed primarily in skeletal muscle. UCP3 is upregulated in various conditions characterized by skeletal muscle atrophy, including hyperthyroidism, fasting, denervation, diabetes, cancer, lipopolysaccharide (LPS), and treatment with glucocorticoids (GCs). The influence of sepsis, another condition characterized by muscle cachexia, on UCP3 expression and activity is not known. We examined UCP3 gene and protein expression in skeletal muscles from rats after cecal ligation and puncture and from sham-operated control rats. Sepsis resulted in a two- to threefold increase in both mRNA and protein levels of UCP3 in skeletal muscle. Treatment of rats with the glucocorticoid receptor antagonist RU-38486 prevented the sepsis-induced increase in gene and protein expression of UCP3. The UCP3 mRNA and protein levels were increased 2.4- to 3.6-fold when incubated muscles from normal rats were treated with dexamethasone (DEX) and/or free fatty acids (FFA ) ex vivo. In addition, UCP3 mRNA and protein levels were significantly increased in normal rat muscles in vivo with treatment of either DEX or FFA. The results suggest that sepsis upregulates the gene and protein expression of UCP3 in skeletal muscle, which may at least in part be mediated by GCs and FFA.
Dual effects on HDL metabolism by cholesteryl ester transfer protein inhibition in HepG2 cells
Authors: Huang Zhiping , Inazu Akihiro , Kawashiri Masa-aki , Nohara Atsushi , Higashikata Toshinori , Mabuchi Hiroshi
Published: March 2015

Cholesteryl ester transfer protein (CETP) promotes reverse cholesterol transport via exchange of cholesteryl ester and triglyceride among lipoproteins. Here, we focused on HDL metabolism during inhibition of CETP expression by using CETP antisense oligodeoxynucleotides (ODNs) in HepG2 cells. CETP secretion was decreased by 70% in mRNA levels and by 52% in mass 20 h after ODNs against CETP were delivered to HepG2 cells. Furthermore, as a consequence of the downregulation of CETP, the expression of scavenger receptor class B type I (SR-BI), an HDL receptor, was also reduced by ∼50% in mRNA and protein levels, whereas the apolipoprotein A-I (apoA-I) expression and secretion were increased by 30 and 92%, respectively. In a functional study, the selective uptake of125I-[14C]cholesteryl oleate-labeled HDL3was decreased. Cholesterol efflux to apoA-I and HDL3was significantly increased by 88 and 37%, respectively. Moreover, the CE levels in cells after antisense treatment were elevated by 20%, which was related to the about twofold increase of cholesterol esterification and increased acyl-CoA:cholesterol acyltransferase 1 mRNA levels. Taken together, these findings suggest that although acute suppression of CETP expression leads to an elevation in cellular cholesterol stores, apoA-I secretion, and cellular cholesterol efflux to apoA-I, the return of HDL-CE to hepatocytes via an SR-BI pathway was inhibited in vitro. Thus antisense inhibition of hepatic CETP expression manifests dual effects: namely, increased formation of HDL and suppression of catabolism of HDL-CE, probably via the SR-BI pathway.
Measurement of protein metabolism in epidermis and dermis
Authors: Zhang Xiao-Jun , Chinkes David L. , Wolfe Robert R.
Published: March 2015

We found that, in the rabbit ear, the dermal protein contains 75.5% of cutaneous phenylalanine and 97.9% of cutaneous proline; the remaining 24.5% of phenylalanine and 2.1% of proline are in the epidermal protein. This finding led us to develop two novel models that use phenylalanine and proline tracers and the rabbit ear to quantify protein kinetics in the epidermis and dermis. The four-pool model calculates the absolute rates of protein kinetics and amino acid transport, and the two-pool model calculates the apparent rates of protein kinetics that are reflected in the blood. The results showed that both epidermis and dermis maintained their protein mass in the postabsorptive state. The rate of epidermal protein synthesis was 93.4 ± 37.6 mg · 100 g−1 · h−1, which was 10-fold greater than that of the dermal protein (9.3 ± 5.8 mg · 100 g−1 · h−1). These synthetic rates were in agreement with those measured simultaneously by the tracer incorporation method. Comparison of the four-pool and two-pool models indicated that intracellular cycling of amino acids accounted for 75 and 90% of protein kinetics in the dermis and epidermis, respectively. We conclude that, in the skin, efficient reutilization of amino acids from proteolysis for synthesis enables the maintenance of protein mass in the postabsorptive state.
Potentiation of insulin secretion by phorbol esters is mediated by PKC-α and nPKC isoforms
Authors: Yaney Gordon C. , Fairbanks Jamison M. , Deeney Jude T. , Korchak Helen M. , Tornheim Keith , Corkey Barbara E.
Published: March 2015

Culturing clonal β-cells (HIT-T15) overnight in the presence of phorbol ester [phorbol myristate acetate (PMA)] enhanced insulin secretion while causing downregulation of some protein kinase C (PKC) isoforms and most PKC activity. We show here that this enhanced secretion required the retention of PMA in the cell. Hence, it could not be because of long-lived phosphorylation of cellular substrates by the isoforms that were downregulated, namely PKC-α, -βII, and -ε, but could be because of the continued activation of the two remaining diacylglycerol-sensitive isoforms δ and μ. The enhanced secretion did not involve changes in glucose metabolism, cell membrane potential, or intracellular Ca2+handling, suggesting a distal effect. PMA washout caused the loss of the enhanced response, but secretion was then stimulated by acute readdition of PMA or bombesin. The magnitude of this restimulation appeared dependent on the mass of PKC-α, which was rapidly resynthesized during PMA washout. Therefore, stimulation of insulin secretion by PMA, and presumably by endogenous diacylglycerol, involves the activation of PKC isoforms δ and/or μ, and also PKC-α.
Human placenta metabolizes fatty acids: implications for fetal fatty acid oxidation disorders and maternal liver diseases
Authors: Shekhawat Prem , Bennett Michael J. , Sadovsky Yoel , Nelson D. Michael , Rakheja Dinesh , Strauss Arnold W.
Published: March 2015

The role of fat metabolism during human pregnancy and in placental growth and function is poorly understood. Mitochondrial fatty acid oxidation disorders in an affected fetus are associated with maternal diseases of pregnancy, including preeclampsia, acute fatty liver of pregnancy, and the hemolysis, elevated liver enzymes, and low platelets syndrome called HELLP. We have investigated the developmental expression and activity of six fatty acid β-oxidation enzymes at various gestational-age human placentas. Placental specimens exhibited abundant expression of all six enzymes, as assessed by immunohistochemical and immunoblot analyses, with greater staining in syncytiotrophoblasts compared with other placental cell types. β-Oxidation enzyme activities in placental tissues were higher early in gestation and lower near term. Trophoblast cells in culture oxidized tritium-labeled palmitate and myristate in substantial amounts, indicating that the human placenta utilizes fatty acids as a significant metabolic fuel. Thus human placenta derives energy from fatty acid oxidation, providing a potential explanation for the association of fetal fatty acid oxidation disorders with maternal liver diseases in pregnancy.
Acute cold exposure, leptin, and somatostatin analog (octreotide) modulate thyroid 5′-deiodinase activity
Authors: Lisboa Patricia Cristina , Oliveira Karen Jesus , Cabanelas Adriana , Ortiga-Carvalho Tania Maria , Pazos-Moura Carmen Cabanelas
Published: March 2015

We investigated the effect of acute cold exposure, leptin, and the somatostatin analog octreotide (OCT) on thyroid type I (D1) and II (D2) deiodinase activities. Microsomal D1 and D2 activities were measured by the release of 125I from 125I-reverse triiodothyronine (rT3) under different assay conditions. Rats exposed to 4°C (15, 30, 60, and 120 min) showed progressive reduction in thyroidal D1 and D2, reaching ∼40% at 2 h ( P < 0.05) despite increased circulating TSH ( P < 0,05) associated with the higher thyroid D1 and D2 in hypothyroid rats. A single injection of leptin (8 μg/100 g body wt sc) induced increased thyroid and liver D1 ( P < 0.05), but not thyroid D2, activities at 30 and 120 min, independently of the serum TSH rise shown only at 2 h. OCT (1 μg/kg body wt sc) increased D1 and D2 activity significantly 24 h after a single injection, with no changes in serum TSH. Therefore, leptin and somatostatin are potential physiological upregulators of thyroid deiodinases, and their low secretion during acute cold exposure may be a potential mechanism contributing to cold-induced reduction in thyroid deiodinase activity.
Myocardial interstitial glucose and lactate before, during, and after cardioplegic heart arrest
Authors: Kennergren Charles , Mantovani Vittorio , Strindberg Lena , Berglin Eva , Hamberger Anders , Lönnroth Peter
Published: March 2015

The interstitial fluid of the human myocardium was monitored in 13 patients undergoing aortic valve and/or bypass surgery before, during, and after hypothermic potassium cardioplegia. The regulation of glucose and lactate was studied after sampling with microdialysis. The following questions were addressed. 1) Is the rate of transcapillary diffusion the limiting step for myocardial uptake of glucose before or after cardioplegia? 2) Does cold potassium cardioplegia induce a critical deprivation of glucose and/or accumulation of lactate in the myocardium? Before cardioplegia, interstitial glucose was ∼50% of the plasma level ( P < 0.001). Interstitial glucose decreased significantly immediately after induction of cardioplegia and remained low (1.25 ± 0.25 mM) throughout cardioplegia. It was restored to precardioplegic levels 1 h after release of the aortic clamp. Interstitial glucose then decreased again at 25 and 35 h postoperatively to the levels observed during cardioplegia. Interstitial lactate decreased immediately after induction of cardioplegia but returned to basal level during the clamping period. At 25 and 35 h, interstitial lactate was significantly lower than before and during cardioplegia. Glucose transport over the capillary endothelium is considered rate limiting for its uptake in the working heart but not during cold potassium cardioplegia despite the glucose deprivation following perfusion of glucose-free cardioplegic solution. Lactate accumulated during cardioplegia but never reached exceedingly high interstitial levels. We conclude that microdialysis provides information that may be relevant for myocardial protection during open-heart surgery.
Testosterone administration to men increases hepatic lipase activity and decreases HDL and LDL size in 3 wk
Authors: Herbst Karen L. , Amory John K. , Brunzell John D. , Chansky Howard A. , Bremner William J.
Published: March 2015

Testosterone administration to men is known to decrease high-density lipoprotein cholesterol (HDL-C) and the subclasses HDL2and HDL3. It also might increase the number of small, dense, low-density lipoprotein cholesterol (LDL-C) particles in hypogonadal men. The decrease in HDL-C and in LDL-C size is potentially mediated by hepatic lipase activity, which hydrolyzes lipoprotein phospholipids and triacylglycerol. To determine how HDL-C and LDL-C particles are affected by testosterone administration to eugonadal men, testosterone was administered as a supraphysiological dose (600 mg/wk) for 3 wk to elderly, obese, eugonadal men before elective hip or knee surgery, and lipids were measured by routine methods and by density gradient ultracentrifugation. Hepatic lipase activity increased >60% above baseline levels, and HDL-C, HDL2, and HDL3significantly declined in 3 wk. In addition, the LDL-C peak particle density and the amount of LDL-C significantly increased. Testosterone is therefore a potent stimulator of hepatic lipase activity, decreasing HDL-C, HDL2, and HDL3as well as increasing LDL particle density changes, all associated with increased cardiovascular risk.
Characterization of MCH-mediated obesity in mice
Authors: Ito Masahiko , Gomori Akira , Ishihara Akane , Oda Zenjun , Mashiko Satoshi , Matsushita Hiroko , Yumoto Mariko , Ito Makoto , Sano Hideki , Tokita Shigeru , Moriya Minoru , Iwaasa Hisashi , Kanatani Akio
Published: March 2015

Melanin-concentrating hormone (MCH) is a cyclic orexigenic peptide expressed in the lateral hypothalamus. Recently, we demonstrated that chronic intracerebroventricular infusion of MCH induced obesity accompanied by sustained hyperphagia in mice. Here, we analyzed the mechanism of MCH-induced obesity by comparing animals fed ad libitum with pair-fed and control animals. Chronic infusion of MCH significantly increased food intake, body weight, white adipose tissue (WAT) mass, and liver mass in ad libitum-fed mice on a moderately high-fat diet. In addition, a significant increase in lipogenic activity was observed in the WAT of the ad libitum-fed group. Although body weight gain was marginal in the pair-fed group, MCH infusion clearly enhanced the lipogenic activity in liver and WAT. Plasma leptin levels were also increased in the pair-fed group. Furthermore, MCH infusion significantly reduced rectal temperatures in the pair-fed group. In support of these findings, mRNA expression of uncoupling protein-1, acyl-CoA oxidase, and carnitine palmitoyltransferase I, which are key molecules involved in thermogenesis and fatty acid oxidation, were reduced in the brown adipose tissue (BAT) of the pair-fed group, suggesting that MCH infusion might reduce BAT functions. We conclude that the activation of MCH neuronal pathways stimulated adiposity, in part resulting from increased lipogenesis in liver and WAT and reduced energy expenditure in BAT. These findings confirm that modulation of energy homeostasis by MCH may play a critical role in the development of obesity.
Protein acylation in the inhibition of insulin secretion by norepinephrine, somatostatin, galanin, and PGE2
Authors: Cheng Haiying , Straub Susanne G. , Sharp Geoffrey W. G.
Published: March 2015

The major physiological inhibitors of insulin secretion, norepinephrine, somatostatin, galanin, and prostaglandin E2, act via specific receptors that activate pertussis toxin (PTX)-sensitive G proteins. Four inhibitory mechanisms are known: 1) activation of ATP-sensitive K channels and repolarization of the β-cell; 2) inhibition of L-type Ca2+channels; 3) decreased activity of adenylyl cyclase; and 4) inhibition of exocytosis at a “distal” site in stimulus-secretion coupling. We have examined the underlying mechanisms of inhibition at this distal site. In rat pancreatic islets, 2-bromopalmitate, cerulenin, and polyunsaturated fatty acids, all of which suppress protein acyltransferase activity, blocked the distal inhibitory effects of norepinephrine in a concentration-dependent manner. In contrast, control compounds such as palmitate, 16-hydroxypalmitate, and etomoxir, which do not block protein acylation, had no effect. Furthermore, 2-bromopalmitate also blocked the distal inhibitory actions of somatostatin, galanin, and prostaglandin E2. Importantly, neither 2-bromopalmitate nor cerulenin affected the action of norepinephrine to decrease cAMP production. We also examined the effects of norepinephrine, 2-bromopalmitate, and cerulenin on palmitate metabolism. Palmitate oxidation and its incorporation into lipids seemed not to contribute to the effects of 2-bromopalmitate and cerulenin on norepinephrine action. These data suggest that protein acylation mediates the distal inhibitory effect on insulin secretion. We propose that the inhibitors of insulin secretion, acting via PTX-sensitive G proteins, activate a specific protein acyltransferase, causing the acylation of a protein or proteins critical to exocytosis. This particular acylation and subsequent disruption of the essential and precise interactions involved in core complex formation would block exocytosis.
Involvement of the vagus nerves in the regulation of basal hepatic glucose production in conscious dogs
Authors: Cardin Sylvain , Walmsley Konstantin , Neal Doss W. , Williams Phillip E. , Cherrington Alan D.
Published: March 2015

We determined if blocking transmission in the fibers of the vagus nerves would affect basal hepatic glucose metabolism in the 18-h-fasted conscious dog. A pancreatic clamp (somatostatin, basal portal insulin, and glucagon) was employed. A 40-min control period was followed by a 90-min test period. In one group, stainless steel cooling coils (Sham, n = 5) were perfused with a 37°C solution, while in the other (Cool, n = 6), the coils were perfused with −20°C solution. Vagal blockade was verified by heart rate change (80 ± 9 to 84 ± 14 beats/min in Sham; 98 ± 12 to 193 ± 22 beats/min in Cool). The arterial glucose level was kept euglycemic by glucose infusion. No change in tracer-determined glucose production occurred in Sham, whereas in Cool it dropped significantly (2.4 ± 0.4 to 1.9 ± 0.4 mg · kg−1· min−1). Net hepatic glucose output did not change in Sham but decreased from 1.9 ± 0.3 to 1.3 ± 0.3 mg · kg−1· min−1in the Cool group. Hepatic gluconeogenesis did not change in either group. These data suggest that vagal blockade acutely modulates hepatic glucose production by inhibiting glycogenolysis.
The influence of insulin on circulating ghrelin
Authors: Flanagan Daniel E. , Evans Mark L. , Monsod Teresa P. , Rife Frances , Heptulla Rubina A. , Tamborlane William V. , Sherwin Robert S.
Published: March 2015

Ghrelin is a novel peptide that acts on the growth hormone (GH) secretagogue receptor in the pituitary and hypothalamus. It may function as a third physiological regulator of GH secretion, along with GH-releasing hormone and somatostatin. In addition to the action of ghrelin on the GH axis, it appears to have a role in the determination of energy homeostasis. Although feeding suppresses ghrelin production and fasting stimulates ghrelin release, the underlying mechanisms controlling this process remain unclear. The purpose of this study was to test the hypotheses, by use of a stepped hyperinsulinemic eu- hypo- hyperglycemic glucose clamp, that either hyperinsulinemia or hypoglycemia may influence ghrelin production. Having been stable in the period before the clamp, ghrelin levels rapidly fell in response to insulin infusion during euglycemia (baseline ghrelin 207 ± 12 vs. 169 ± 10 fmol/ml at t = 30 min, P < 0.001). Ghrelin remained suppressed during subsequent periods of hypoglycemia (mean glucose 53 ± 2 mg/dl) and hyperglycemia (mean glucose 163 ± 6 mg/dl). Despite suppression of ghrelin, GH showed a significant rise during hypoglycemia (baseline 4.1 ± 1.3 vs. 28.2 ± 3.9 μg/l at t = 120 min, P < 0.001). Our data suggest that insulin may suppress circulating ghrelin independently of glucose, although glucose may have an additional effect. We conclude that the GH response seen during hypoglycemia is not regulated by circulating ghrelin.
Normalization of growth in hypophysectomized mice using hydrodynamic transfer of the human growth hormone gene
Authors: Søndergaard Morten , Dagnæs-Hansen Frederik , Flyvbjerg Allan , Jensen Thomas G.
Published: March 2015

Nonviral gene transfer was investigated as a potential treatment of growth hormone deficiency (GHD) using hypophysectomized mice as a model. After a single hydrodynamic administration of naked plasmid DNA containing the human growth hormone (hGH) gene controlled by an ubiquitin promoter, sustained elevation of circulating hGH was observed the entire observation period (68 days), with a concomitant normalization of circulating insulin-like growth factor I (IGF-I) and IGF-binding protein-3. Furthermore, longitudinal growth was corrected in terms of normalization of tibia length, tail length, and body weight gain. Liver, spleen, and lung weights were normalized, whereas heart weight was normalized partly. hGH mRNA was expressed exclusively in liver tissue. In conclusion, we showed that nonviral hGH gene transfer normalizes longitudinal growth in hypophysectomized mice, indicating that this method potentially could be relevant as a new therapeutic tool in the clinical handling of GHD.
Reduction of spontaneous and irradiation-induced apoptosis in small intestine of IGF-I transgenic mice
Authors: Wilkins Heather R. , Ohneda Kinuko , Keku Temitope O. , D'Ercole A. Joseph , Fuller C. Randall , Williams Kristen L. , Lund P. Kay
Published: March 2015

Insulin-like growth factor I (IGF-I) may promote survival of putative stem cells in the small intestinal epithelium. Mitosis and apoptosis were quantified in crypts of nonirradiated and irradiated IGF-I transgenic (TG) and wild-type (WT) littermates. The mean apoptotic index was significantly greater in WT vs. TG littermates. After irradiation, apoptotic indexes increased, and WT mice showed a more dramatic increase in apoptosis than TG mice at the location of putative stem cells. After irradiation, no mitotic figures were observed in WT crypts, whereas mitosis was maintained within the jejunal epithelium of TG mice. The abundance and localization of Bax mRNA did not differ between nonirradiated littermates. However, there was more Bax mRNA in TG vs. WT mice after irradiation. Bax mRNA was located along the entire length of the irradiated crypt epithelium, but there was less Bax protein observed in the bottom third of TG mouse crypts compared with WT littermates. IGF-I regulates cell number by stimulating crypt cell proliferation and decreasing apoptosis preferentially within the stem cell compartment.
Prolonged infusion of angiotensin II into normal rats induces stellate cell activation and proinflammatory events in liver
Authors: Bataller Ramón , Gäbele Erwin , Schoonhoven Robert , Morris Terry , Lehnert Mark , Yang Liu , Brenner David A. , Rippe Richard A.
Published: March 2015

Recent evidence indicates that angiotensin II (ANG II) plays an important role in liver fibrogenesis. However, the underlying mechanisms are largely unknown. In advanced chronic liver diseases, circulating levels of ANG II are frequently elevated. We investigated the hepatic effects of prolonged systemic infusion of ANG II in normal rats. Saline or ANG II at subpressor and pressor doses (15 and 50 ng·kg-1·min-1, respectively) were infused to normal rats for 4 wk through a subcutaneous osmotic pump. Infusion of ANG II resulted in liver injury, as assessed by elevated serum liver enzymes. Livers from ANG II-perfused rats showed activation of JNK and ERK as well as increased NF-κB and activating protein-1 DNA-binding activity. Moreover, ANG II perfusion induced oxidative stress, increased concentration of proinflammatory cytokines, and upregulated the inflammatory proteins inducible nitric oxide synthase and cyclooxygenase-2. Histological examination of the livers from ANG II-infused rats showed mild portal inflammation as well as thickening and thrombosis of small hepatic vessels. ANG II-treated livers showed accumulation of CD43-positive inflammatory cells and activated hepatic stellate cells (HSCs) at the pericentral areas. A slight increase in collagen synthesis was observed, as assessed by Sirius red staining and hepatic hydroxyproline. All of these effects were observed when ANG II was perfused at subpressor and pressor doses. ANG II also accelerated the activation of primary cultured rat HSCs. In conclusion, increased systemic ANG II can induce liver injury by promoting proinflammatory events and vascular damage. ANG II-induced hepatic effects are not dependent on increase in arterial pressure.
Antiapoptotic effect of EGF on mouse hepatocytes associated with downregulation of proapoptotic Bid protein
Authors: Éthier Chantal , Raymond Valérie-Ann , Musallam Lina , Houle Robert , Bilodeau Marc
Published: March 2015

Growth factors have been shown to protect cells from a variety of apoptotic stimuli. In the liver, the Fas system is thought to be very important in the genesis of hepatocyte apoptosis. Others have already shown the importance of the phosphatidylinositol 3-kinase (PI3-kinase) pathway and of increased Bcl-xl expression in the antiapoptotic effect of growth factors on hepatocytes. We investigated the effect of EGF on Bid, a BH3-only member of the Bcl-2 family and a major player in the transduction of the Fas apoptotic signal. Hepatocyte apoptosis was induced in vitro with a purified anti-mouse Fas antibody. The effect of EGF on Bid protein expression was studied on those cultures. EGF dose dependently reduced the expression of Bid protein in primary mouse hepatocyte cultures independently of Fas stimulation. This decrease was not the result of the degradation of Bid into its active p15 fragment. Treating cells with a specific inhibitor of the EGF receptor autophosphorylation completely abolished the decrease in Bid expression afforded by EGF. Treatment with LY-294002, a PI3-kinase blocker, partly reverted the effect of EGF. When apoptosis was induced in Bid-deficient hepatocytes, EGF lost its capacity to protect cells against this type of cell death. These results show that EGF decreases the expression of Bid protein and suggest that the effect of EGF on Bid is one of the mechanisms of the antiapoptotic effect of EGF.
III. Imaging and the gastrointestinal tract: mapping the human enteric nervous system
Authors: Schemann Michael , Michel Klaus , Peters Saskia , Bischoff Stephan C. , Neunlist Michel
Published: March 2015

Monitoring membrane potentials by multisite optical recording techniques using voltage-sensitive dyes is ideal for direct analysis of network signaling. We applied this technology to monitor fast and slow excitability changes in the enteric nervous system and in hundreds of neurons simultaneously at cellular and subcellular resolution. This imaging technique presents a powerful tool to study activity patterns in enteric pathways and to assess differential activation of nerves in the gut to a number of stimuli that modulate neuronal activity directly or through synaptic mechanisms. The optical mapping made it possible to record from tissues such as human enteric nerves, which were, until now, inaccessible by other techniques.
Regulation of IL-11 expression in intestinal myofibroblasts: role of c-Jun AP-1- and MAPK-dependent pathways
Authors: Bamba Shigeki , Andoh Akira , Yasui Hirofumi , Makino Jin , Kim Shokei , Fujiyama Yoshihide
Published: March 2015

IL-11 inhibits the activation of NF-κB and induces the Th2 polarization of CD4+T cells. The clinical utility of IL-11 is being investigated in Crohn's disease. However, physiological secretion of IL-11 in the intestine remains unclear. In this study, we investigated IL-11 secretion in human intestinal subepithelial myofibroblasts (SEMFs). Intestinal SEMFs were isolated from the human colonic mucosa. IL-11 secretion and mRNA expression were determined by ELISA and Northern blot analysis. The activating protein (AP)-1-DNA binding activity was evaluated by EMSA. IL-11 secretion was induced by IL-1β and transforming growth factor (TGF)-β1. These were also observed at the mRNA level. The EMSAs demonstrated that both IL-1β and TGF-β1 induced AP-1 activation within 2 h after stimulation, and a blockade of AP-1 activation by the recombinant adenovirus containing a dominant negative c-Jun markedly reduced the IL-1β- and TGF-β1-induced IL-11 mRNA expression. IL-1β and TGF-β1 induced an activation of ERK p42/44 and p38 MAP kinases, and the MAP kinase inhibitors (SB-202190, PD-98059, and U-0216) significantly reduced the IL-1β- and TGF-β1-induced IL-11 secretion. The upregulation of IL-11 mRNA by IL-1β- and TGF-β1 was also mediated by a p38 MAP kinase-mediated mRNA stabilization. The combination of IL-1β and TGF-β1 additively enhanced IL-11 secretion. Intestinal SEMFs secreted IL-11 in response to IL-1β- and TGF-β1. Mucosal IL-11 secretion might be important as an anti-inflammatory response in the pathogenesis of intestinal inflammation.
Muscarinic receptors couple to modulation of nicotinic ACh receptor desensitization in myenteric neurons
Authors: Brown Erika N. , Galligan James J.
Published: March 2015

Signaling mechanisms coupled to activation of different neurotransmitter receptors interact in the enteric nervous system. ACh excites myenteric neurons by activating nicotinic ACh receptors (nAChRs) and muscarinic receptors expressed by the same neurons. These studies tested the hypothesis that muscarinic receptor activation alters the functional properties of nAChRs in guinea pig small intestinal myenteric neurons maintained in primary culture. Whole cell patch-clamp techniques were used to measure inward currents caused by ACh (1 mM) or nicotine (1 mM). Currents caused by ACh and nicotine were blocked by hexamethonium (100 μM) and showed complete cross desensitization. The rate and extent of nAChR desensitization was greater when recordings were obtained with ATP/GTP-containing compared with ATP/GTP-free pipette solutions. These data suggest that ATP/GTP-dependent mechanisms increase nAChR desensitization. The muscarinic receptor antagonist scopolamine (1 μM) decreased desensitization caused by ACh but not by nicotine, which does not activate muscarinic receptors. Phorbol 12,13-dibutyrate (10–100 nM), an activator of protein kinase C (PKC), but not 4-α-phorbol 12-myristate 13-acetate (a PKC inactive phorbol ester), increased nAChR desensitization caused by ACh and nicotine. Forskolin (1 μM), an activator of adenylate cyclase, increased nAChR desensitization, but this effect was mimicked by dideoxyforskolin, an adenylate cyclase inactive forskolin analog. These data indicate that simultaneous activation of nAChRs and muscarinic receptors increases nAChR desensitization. This effect may involve activation of a PKC-dependent pathway. These data also suggest that nAChRs and muscarinic receptors are coupled functionally through an intracellular signaling pathway in myenteric neurons.
Impulse-response function of splanchnic circulation with model-independent constraints: theory and experimental validation
Authors: Munk Ole L. , Keiding Susanne , Bass Ludvik
Published: March 2015

Modeling physiological processes using tracer kinetic methods requires knowledge of the time course of the tracer concentration in blood supplying the organ. For liver studies, however, inaccessibility of the portal vein makes direct measurement of the hepatic dual-input function impossible in humans. We want to develop a method to predict the portal venous time-activity curve from measurements of an arterial time-activity curve. An impulse-response function based on a continuous distribution of washout constants is developed and validated for the gut. Experiments with simultaneous blood sampling in aorta and portal vein were made in 13 anesthetized pigs following inhalation of intravascular [15O]CO or injections of diffusible 3- O-[11C]methylglucose (MG). The parameters of the impulse-response function have a physiological interpretation in terms of the distribution of washout constants and are mathematically equivalent to the mean transit time ( T̄) and standard deviation of transit times. The results include estimates of mean transit times from the aorta to the portal vein in pigs: T̄ = 0.35 ± 0.05 min for CO and 1.7 ± 0.1 min for MG. The prediction of the portal venous time-activity curve benefits from constraining the regression fits by parameters estimated independently. This is strong evidence for the physiological relevance of the impulse-response function, which includes asymptotically, and thereby justifies kinetically, a useful and simple power law. Similarity between our parameter estimates in pigs and parameter estimates in normal humans suggests that the proposed model can be adapted for use in humans.
Regulation of expression of the intestinal oligopeptide transporter (Pept-1) in health and disease
Authors: Adibi Siamak A.
Published: March 2015

The abundance of the oligopeptide transporter (Pept-1) in the brush-border membrane of the intestinal epithelium is the central mechanism for regulation of transport of products of protein digestion (dipeptides and tripeptides) and peptidomimetic drugs (for example, β-lactam antibiotics). Within the past few years, there has been substantial progress in identifying the factors controlling this regulation and the mechanisms of their actions. The purpose of this report is to review this progress. The studies of individual substrates and hormones in a human intestinal cell line (Caco-2) have shown that dipeptides, certain amino acids, insulin, and leptin increase and epidermal growth factor and triiodothyronine decrease the membrane population of Pept-1. In the case of dipeptides, epidermal growth factor, and thyroid hormone, there are parallel changes in the gene expression brought about by alteration of transcription and/or stability of Pept-1 mRNA. In contrast, the treatment with insulin and leptin does not induce any alteration in the Pept-1 gene expression, and the mechanism of increased protein expression appears to be increased trafficking from a preformed cytoplasmic pool to the apical membrane. In vivo studies in rats have shown modulation of protein and gene expressions of the intestinal oligopeptide transporter during the day and during development and in nutritional and metabolic alterations, such as high-protein diet, fasting, and diabetes. Patients with intestinal diseases, such as ulcerative colitis, Crohn's disease, and short-bowel syndrome, may have induction of the Pept-1 expression in their colon. Finally, pharmacological studies have shown that the expression of Pept-1 can be upregulated by agents such as 5 fluorouracil and downregulated by agents such as cyclosporine. In conclusion, the above studies have produced a wealth of new information on regulation of a key transporter in the intestine. This information may have useful applications in nutritional and pharmacological treatments, for example, in diabetic patients needing enteral nutrition or in ulcerative colitis patients needing the suppression of the intestinal inflammation.
Biotin uptake by human intestinal and liver epithelial cells: role of the SMVT system
Authors: Balamurugan Krishnaswamy , Ortiz Alvaro , Said Hamid M.
Published: March 2015

It has been well established that human intestinal and liver epithelial cells transport biotin via an Na+-dependent carrier-mediated mechanism. The sodium-dependent multivitamin transport (SMVT), a biotin transporter, is expressed in both cell types. However, the relative contribution of SMVT toward total carrier-mediated uptake of physiological (nanomolar) concentrations of biotin by these cells is not clear. Addressing this issue is important, especially in light of the recent identification of a second human high-affinity biotin uptake mechanism that operates at the nanomolar range. Hence, we employed a physiological approach of characterizing biotin uptake by human-derived intestinal Caco-2 and HepG2 cells at the nanomolar concentration range. We also employed a molecular biology approach of selectively silencing the endogenous SMVT of these cells with specific small interfering RNAs (siRNAs), then examining carrier-mediated biotin uptake. The results showed that in both Caco-2 and HepG2 cells, the initial rate of biotin uptake as a function of concentration over the range of 0.1 to 50 nM to be linear. Furthermore, we found that the addition of 100 nM unlabeled biotin, desthiobiotin, or pantothenic acid to the incubation medium had no effect on the uptake of 2.6 nM [3H]biotin. Pretreatment of Caco-2 and HepG2 cells with SMVT specific siRNAs substantially reduced SMVT mRNA and protein levels. In addition, carrier-mediated [3H]biotin (2.6 nM) uptake by Caco-2 and HepG2 cells was severely ( P 0.01) inhibited by the siRNAs pretreatment. These results demonstrate that the recently described human high-affinity biotin uptake system is not functional in intestinal and liver epithelial cells. In addition, the results provide strong evidence that SMVT is the major (if not the only) biotin uptake system that operates in these cells.
α-Melanocyte-stimulating hormone protects against mesenteric ischemia-reperfusion injury
Authors: Hassoun Heitham T. , Zou Lei , Moore Frederick A. , Kozar Rosemary A. , Weisbrodt Norman W. , Kone Bruce C.
Published: March 2015

Mesenteric ischemia-reperfusion (I/R) injury to the intestine is a common and often devastating clinical occurrence for which there are few therapeutic options. α-Melanocyte-stimulating hormone (α-MSH) is a tridecapeptide released by the pituitary gland and immunocompetent cells that exerts anti-inflammatory actions and abrogates postischemic injury to the kidneys and brainstem of rodents. To test the hypothesis that α-MSH would afford similar protection in the postischemic small intestine, we analyzed the effects of this peptide on intestinal transit, histology, myeloperoxidase activity, and nuclear factor-κB (NF-κB) activation after 45 min of superior mesenteric artery occlusion and ≤6 h of reperfusion. Rats subjected to I/R exhibited markedly depressed intestinal transit, histological evidence of severe injury to the ileum, increased myeloperoxidase activity in ileal cytoplasmic extracts, and biphasic activation of NF-κB in ileal nuclear extracts. In contrast, rats treated with α-MSH before I/R exhibited intestinal transit and histological injury scores comparable to those of sham-operated controls. In addition, the α-MSH-treated rats demonstrated less I/R-induced activation of intestinal NF-κB and myeloperoxidase activity after prolonged (6 h) reperfusion. We conclude that α-MSH significantly limits postischemic injury to the rat small intestine.