bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevention of glycogen supercompensation prolongs the increase in muscle GLUT4 after exercise
Authors: Garcia-Roves Pablo M. , Han Dong-Ho , Song Zheng , Jones Terry E. , Hucker Kathleen A. , Holloszy John O.
Published: March 2015

Exercise induces an increase in GLUT4 in skeletal muscle with a proportional increase in glucose transport capacity. This adaptation results in enhanced glycogen accumulation, i.e., “supercompensation,” in response to carbohydrate feeding after glycogen-depleting exercise. The increase in GLUT4 reverses within 40 h after exercise in carbohydrate-fed rats. The purpose of this study was to determine whether prevention of skeletal muscle glycogen supercompensation after exercise results in maintenance of the increases in GLUT4 and the capacity for glycogen supercompensation. Rats were exercised by means of three daily bouts of swimming. GLUT4 mRNA was increased ∼3-fold and GLUT4 protein was increased ∼2-fold 18 h in epitrochlearis muscle after exercise. These increases in GLUT4 mRNA and protein reversed completely within 42 h after exercise in rats fed a high-carbohydrate diet. In contrast, the increases in GLUT4 protein, insulin-stimulated glucose transport, and increased capacity for glycogen supercompensation persisted unchanged for 66 h in rats fed a carbohydrate-free diet that prevented glycogen supercompensation after exercise. GLUT4 mRNA was still elevated at 42 h but had returned to baseline by 66 h after exercise in rats fed the carbohydrate-free diet. Glycogen-depleted rats fed carbohydrate 66 h after exercise underwent muscle glycogen supercompensation with concomitant reversal of the increase in GLUT4. These findings provide evidence that prevention of glycogen supercompensation after exercise results in persistence of exercise-induced increases in GLUT4 protein and enhanced capacity for glycogen supercompensation.
Differential transcriptional activation of select metabolic genes in response to variations in exercise intensity and duration
Authors: Hildebrandt Audrey L. , Pilegaard Henriette , Neufer P. Darrell
Published: March 2015

Cellular adaptations to endurance training are influenced by the intensity and duration of exercise. To examine the impact of exercise intensity and duration on the acute transcriptional regulation of metabolic genes in red (RG) and white (WG) gastrocnemius muscle, rats completed either low-intensity [∼50% maximal O2uptake (V̇o2max)] treadmill exercise (LIE) for 45 min, LIE for 180 min, or high-intensity (∼75% V̇o2max) exercise (HIE) for 45 min. LIE for 45 min activated ( P < 0.05) transcription of the pyruvate dehydrogenase kinase-4 (PDK4), uncoupling protein-3 (UCP3), heme oxygenase-1 (HO-1), and hexokinase II (HK II) genes in RG within 1 h after exercise. In WG, transcription of PDK4, UCP3, HKII, and lipoprotein lipase (LPL) was also induced, whereas transcription of the HO-1 gene did not change. In RG, extending LIE duration from 45 to 180 min elicited a similar activation of PDK4 and UCP3 (∼15-fold) but a far greater increase in HO-1 (>30-fold) and HKII transcription (>25-fold). In WG, extending LIE for 180 min induced a much greater and prolonged (through 2- to 4-h recovery) activation of PDK4, UCP3 (both >200-fold), and HO-1 (>10-fold). HIE elicited a similar pattern of gene activation to LIE in both RG and WG, with the exception that HIE triggered >10-fold activation of HO-1 in WG. These data provide evidence that both the intensity and the duration of exercise affect the transcriptional regulation of metabolic genes in muscle in a fiber type-specific manner, possibly reflecting the relative stress imposed by the exercise bout.
24-Hydroxylase: potential key regulator in hypervitaminosis D3in growing dogs
Authors: Tryfonidou M. A. , Oosterlaken-Dijksterhuis M. A. , Mol J. A. , van den Ingh T. S. G. A. M. , van den Brom W. E. , Hazewinkel H. A. W.
Published: March 2015

A group of growing dogs supplemented with cholecalciferol (vitamin D3; HVitD) was studied vs. a control group (CVitD; 54,000 vs. 470 IU vitamin D3/kg diet, respectively) from 3 to 21 wk of age. There were no differences in plasma levels of Piand growth-regulating hormones between groups and no signs of vitamin D3intoxication in HVitD. For the duration of the study in HVitD vs. CVitD, plasma 25-hydroxycholecalciferol levels increased 30- to 75-fold; plasma 24,25-dihydroxycholecalciferol levels increased 12- to 16-fold and were accompanied by increased renal 24-hydroxylase gene expression, indicating increased renal 24-hydroxylase activity. Although the synthesis of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] was increased in HVitD vs. CVitD (demonstrated by [3H]1,25(OH)2D3and increased renal 1α-hydroxylase gene expression), plasma 1,25(OH)2D3levels decreased by 40% as a result of the even more increased metabolic clearance of 1,25(OH)2D3(demonstrated by [3H]1,25(OH)2D3and increased gene expression of intestinal and renal 24-hydroxylase). A shift of the Ca set point for parathyroid hormone to the left indicated increased sensitivity of the chief cells. Effective counterbalance was provided by hypoparathyroidism, hypercalcitoninism, and the key regulator 24-hydroxylase, preventing the development of vitamin D3toxicosis.
Thyroid hormone production rates in rat liver and intestine in vivo: a novel graph theory and experimental solution
Authors: Nguyen Thuvan T. , Mol Koen A. , DiStefano Joseph J.
Published: March 2015

We develop a novel method for finding sufficient experimental conditions for discriminating and quantifying individual biomolecule production sources in distributed, inhomogeneous multisource systems in vivo, and we apply it experimentally to a complex, unsolved problem in endocrinology. The majority of hormonal triiodothyronine (T3) is produced from prohormone thyroxine (T4) in numerous nonthyroidal organs and, with one exception, the T3production rate has not been fully resolved in any single extrathyroidal organ of any species. Using a readily generalized graphic method called cut-set analysis, we show here that measured steady-state responses in several organs to three independent tracer infusions, two into blood and one directly into the organ(s) of interest, are sufficient to resolve this problem for organs fully accessible to direct infusion in vivo. We evaluated local T3production in rat liver and intestine, which also required T3bile flux measurements, and we found that liver produces ∼31% and whole intestine ∼6% of whole body T3from T4. With thyroidal production included, liver contributes ∼15% and intestine ∼3% of whole rat T3production. This new methodology is broadly applicable, especially to biosystems that include molecular interconversions at multiple sites.
Intermittent increases in cytosolic Ca2+stimulate mitochondrial biogenesis in muscle cells
Authors: Ojuka Edward O. , Jones Terry E. , Han Dong-Ho , Chen May , Wamhoff Brian R. , Sturek Michael , Holloszy John O.
Published: March 2015

Muscle contractions cause numerous disturbances in intracellular homeostasis. This makes it impossible to use contracting muscle to identify which of the many signals generated by contractions are responsible for stimulating mitochondrial biogenesis. One purpose of this study was to evaluate the usefulness of L6 myotubes, which do not contract, for studying mitochondrial biogenesis. A second purpose was to evaluate further the possibility that increases in cytosolic Ca2+can stimulate mitochondrial biogenesis. Continuous exposure to 1 μM ionomycin, a Ca2+ionophore, for 5 days induced an increase in mitochondrial enzymes but also caused a loss of myotubes, as reflected in an ∼40% decrease in protein per dish. However, intermittent (5 h/day) exposure to ionomycin, or to caffeine or W7, which release Ca2+from the sarcoplasmic reticulum, did not cause a decrease in protein per dish. Raising cytosolic Ca2+intermittently with these agents induced significant increases in mitochondrial enzymes. EGTA blocked most of this effect of ionomycin, whereas dantrolene, which blocks Ca2+release from the sarcoplasmic reticulum, largely prevented the increases in mitochondrial enzymes induced by W7 and caffeine. These findings provide evidence that intermittently raising cytosolic Ca2+stimulates mitochondrial biogenesis in muscle cells.
Metabolic and cardiorespiratory responses to “the lactate clamp”
Authors: Miller Benjamin F. , Fattor Jill A. , Jacobs Kevin A. , Horning Michael A. , Suh Sang-Hoon , Navazio Franco , Brooks George A.
Published: March 2015

To evaluate the hypothesis that precursor supply limits gluconeogenesis (GNG) during exercise, we examined training-induced changes in glucose kinetics [rates of appearance (Ra) and disappearance (Rd)], oxidation (Rox), and recycling (Rr) with an exogenous lactate infusion to 3.5–4.0 mM during rest and to pretraining 65% peak O2 consumption (V˙o 2 peak) levels during exercise. Control and clamped trials (LC) were performed at rest pre- (PRR, PRR-LC) and posttraining (POR, POR-LC) and during exercise pre- (PREX) and posttraining at absolute (POAB, POAB-LC) and relative (PORL, PORL-LC) intensities. Glucose Rrwas not different in any rest or exercise condition. Glucose Ra did not differ as a result of LC. Glucose Rox was significantly decreased with LC at POR (0.38 ± 0.03 vs. 0.56 ± 0.04 mg · kg−1 · min−1) and POAB (3.82 ± 0.51 vs. 5.0 ± 0.62 mg · kg−1 · min−1). Percent glucose Rd oxidized decreased with all LC except PORL-LC (PRR, 32%; PRR-LC, 22%; POR, 27%; POR-LC, 20%; POAB, 95%; POAB-LC, 77%), which resulted in a significant increase in oxidation from alternative carbohydrate (CHO) sources at rest and POAB. We conclude that 1) increased arterial [lactate] did not increase glucose Rrmeasured during rest or exercise after training, 2) glucose disposal or production did not change with increased precursor supply, and 3) infusion of exogenous CHO in the form of lactate resulted in the decrease of glucose Rox.
Myostatin-deficient mice lose more skeletal muscle mass than wild-type controls during hindlimb suspension
Authors: McMahon Christopher D. , Popovic Ljiljana , Oldham Jenny M. , Jeanplong Ferenc , Smith Heather K. , Kambadur Ravi , Sharma Mridula , Maxwell Linda , Bass James J.
Published: March 2015

Myostatin inhibits myogenesis. Therefore, we sought to determine if mice lacking the myostatin gene [ Mstn(-/-)] would lose less muscle mass than wild-type mice during 7 days of hindlimb suspension (HS). Male Mstn(-/-) and wild-type (C57) mice were subjected to HS or served as ground-based controls ( n = 6/group). Wild-type mice lost 8% of body mass and ∼13% of wet mass from biceps femoris, quadriceps femoris, and soleus, whereas the mass of extensor digitorum longus (EDL) was unchanged after HS. Unexpectedly, Mstn(-/-) mice lost more body (13%, P < 0.05) and quadriceps femoris (17%, P < 0.05) mass than wild-type mice and lost 33% of EDL mass ( P < 0.01) after HS. Protein expression of myostatin in biceps femoris and quadriceps femoris was not altered, whereas expression of MyoD, Myf-5, and myogenin increased in wild-type mice and tended to decrease in muscles of Mstn(-/-) mice. These data suggest that HS induced myogenesis in wild-type mice to counter atrophy, whereas myogenesis was not induced in Mstn(-/-) mice, thereby resulting in a greater loss of muscle mass.
Reversal of denervation-induced insulin resistance by SHIP2 protein synthesis blockade
Authors: Bertelli Daniela F. , Ueno Miriam , Amaral Maria E. C. , Toyama Marcos Hikari , Carneiro Everardo M. , Marangoni Sergio , Carvalho Carla R. O. , Saad Mário J. A. , Velloso Lı́cio A. , Boschero A. Carlos
Published: March 2015

Short-term muscle denervation is a reproducible model of tissue-specific insulin resistance. To investigate the molecular basis of insulin resistance in denervated muscle, the downstream signaling molecules of the insulin-signaling pathway were examined in intact and denervated soleus muscle of rats. Short-term denervation induced a significant fall in glucose clearance rates (62% of control, P < 0.05) as detected by euglycemic hyperinsulinemic clamp and was associated with a significant decrease in insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR; 73% of control, P < 0.05), IR substrate 1 (IRS1; 69% of control, P < 0.05), and IRS2 (82% of control, P < 0.05) and serine phosphorylation of Akt (39% of control, P < 0.05). Moreover, denervation reduced insulin-induced association between IRS1/IRS2 and p85/phosphatidylinositol (PI) 3-kinase. Nevertheless, denervation caused an increase in PI 3-kinase activity associated with IRS1 (275%, P < 0.05) and IRS2 (180%, P < 0.05), but the contents of phosphorylated PI detected by HPLC were significantly reduced in lipid fractions. In the face of the apparent discrepancy, we evaluated the expression and activity of the 5-inositol, lipid phosphatase SH2 domain-containing inositol phosphatase (SHIP2), and the serine phosphorylation of p85/PI 3-kinase. No major differences in SHIP2 expression were detected between intact and denervated muscle. However, serine phosphorylation of p85/PI 3-kinase was reduced in denervated muscle, whereas the blockade of SHIP2 expression by antisense oligonucleotide treatment led to partial restoration of phosphorylated PI contents and to improved glucose uptake. Thus modulation of the functional status of SHIP2 may be a major mechanism of insulin resistance induced by denervation.
Modulation of muscle insulin resistance by selective inhibition of GSK-3 in Zucker diabetic fatty rats
Authors: Henriksen Erik J. , Kinnick Tyson R. , Teachey Mary K. , O'Keefe Matthew P. , Ring David , Johnson Kirk W. , Harrison Stephen D.
Published: March 2015

A role for elevated glycogen synthase kinase-3 (GSK-3) activity in the multifactorial etiology of insulin resistance is now emerging. However, the utility of specific GSK-3 inhibition in modulating insulin resistance of skeletal muscle glucose transport is not yet fully understood. Therefore, we assessed the effects of novel, selective organic inhibitors of GSK-3 (CT-98014 and CT-98023) on glucose transport in insulin-resistant muscles of Zucker diabetic fatty (ZDF) rats. Incubation of type IIb epitrochlearis and type I soleus muscles from ZDF rats with CT-98014 increased glycogen synthase activity (49 and 50%, respectively, P < 0.05) but did not alter basal glucose transport (2-deoxyglucose uptake). In contrast, CT-98014 significantly increased the stimulatory effects of both submaximal and maximal insulin concentrations in epitrochlearis (37 and 24%) and soleus (43 and 26%), and these effects were associated with increased cell-surface GLUT4 protein. Lithium enhanced glycogen synthase activity and both basal and insulin-stimulated glucose transport in muscles from ZDF rats. Acute oral administration (2 × 30 mg/kg) of CT-98023 to ZDF rats caused elevations in GSK-3 inhibitor concentrations in plasma and muscle. The glucose and insulin responses during a subsequent oral glucose tolerance test were reduced by 26 and 34%, respectively, in the GSK-3 inhibitor-treated animals. Thirty minutes after the final GSK-3 inhibitor treatment, insulin-stimulated glucose transport was significantly enhanced in epitrochlearis (57%) and soleus (43%). Two hours after the final treatment, insulin-mediated glucose transport was still significantly elevated (26%) only in the soleus. These results indicate that specific inhibition of GSK-3 enhances insulin action on glucose transport in skeletal muscle of the insulin-resistant ZDF rat. This unique approach may hold promise as a pharmacological treatment against insulin resistance of skeletal muscle glucose disposal.
Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus
Authors: Lechner Andreas , Habener Joel F.
Published: March 2015

In view of the recent success in pancreatic islet transplantation, interest in treating diabetes by the delivery of insulin-producing β-cells has been renewed. Because differentiated pancreatic β-cells cannot be expanded significantly in vitro, β-cell stem or progenitor cells are seen as a potential source for the preparation of transplantable insulin-producing tissue. In addition to embryonic stem (ES) cells, several potential adult islet/β-cell progenitors, derived from pancreas, liver, and bone marrow, are being studied. To date, none of the candidate cells has been fully characterized or is clinically applicable, but pancreatic physiology makes the existence of one or more types of adult islet stem cells very likely. It also seems possible that pluripotential stem cells, derived from the bone marrow, contribute to adult islet neogenesis. In future studies, more stringent criteria should be met to clonally define adult islet/β-cell progenitor cells. If this can be achieved, the utilization of these cells for the generation of insulin-producing β-cells in vitro seems to be feasible in the near future.
Studies with GIP/Ins cells indicate secretion by gut K cells is KATPchannel independent
Authors: Wang Song Yan , Chi Maggie M.-Y. , Li Lin , Moley Kelle H. , Wice Burton M.
Published: March 2015

K cells are a subpopulation of enteroendocrine cells that secrete glucose-dependent insulinotropic polypeptide (GIP), a hormone that promotes glucose homeostasis and obesity. Therefore, it is important to understand how GIP secretion is regulated. GIP-producing (GIP/Ins) cell lines secreted hormones in response to many GIP secretagogues except glucose. In contrast, glyceraldehyde and methyl pyruvate stimulated hormone release. Measurements of intracellular glucose 6-phosphate, fructose 1,6-bisphosphate, and pyruvate levels, as well as glycolytic flux, in glucose-stimulated GIP/Ins cells indicated that glycolysis was not impaired. Analogous results were obtained using glucose-responsive MIN6 insulinoma cells. Citrate levels increased similarly in glucose-treated MIN6 and GIP/Ins cells. Thus pyruvate entered the tricarboxylic acid cycle. Glucose and methyl pyruvate stimulated 1.4- and 1.6-fold increases, respectively, in the ATP-to-ADP ratio in GIP/Ins cells. Glyceraldehyde profoundly reduced, rather than increased, ATP/ADP. Thus nutrient-regulated secretion is independent of the ATP-dependent potassium (KATP) channel. Antibody staining of mouse intestine demonstrated that enteroendocrine cells producing GIP, glucagon-like peptide-1, CCK, or somatostatin do not express detectable levels of inwardly rectifying potassium (Kir) 6.1 or Kir 6.2, indicating that release of these hormones in vivo may also be KATPchannel independent. Conversely, nearly all cells expressing chromogranin A or substance P and ∼50% of the cells expressing secretin or serotonin exhibited Kir 6.2 staining. Compounds that activate calcium mobilization were potent secretagogues for GIP/Ins cells. Secretion was only partially inhibited by verapamil, suggesting that calcium mobilization from intracellular and extracellular sources, independent from KATPchannels, regulates secretion from some, but not all, subpopulations of enteroendocrine cells.
Measurement of TG synthesis and turnover in vivo by2H2O incorporation into the glycerol moiety and application of MIDA
Authors: Turner S. M. , Murphy E. J. , Neese R. A. , Antelo F. , Thomas T. , Agarwal A. , Go C. , Hellerstein M. K.
Published: March 2015

A method is presented for measurement of triglyceride (TG) synthesis that can be applied to slow-turnover lipids. The glycerol moiety of TG is labeled from2H2O, and mass isotopomer distribution analysis (MIDA) is applied. Mice and rats were given 4-8%2H2O in drinking water; TG-glycerol was isolated from adipose and liver during ≤12-wk of2H2O labeling. Mass isotopomer abundances in the glycerol moiety of TG were measured by GC-MS. The combinatorial pattern of isotopomers revealed the number of H atoms in glycerol incorporating label from2H2O (n) to be 3.8–4.0 of a possible 5 for adipose tissue and 4.6–4.8 for liver TG. Hepatic TG-glycerol in fact reached 97% predicted maximal value of label incorporation (4.4–4.6 × body2H2O enrichment), indicating near-complete replacement of the liver TG pool. Label incorporation into adipose tissue revealed turnover of mesenteric TG to be faster ( k = 0.21 day–1) than other depots ( k = 0.04–0.06 day–1) in mice. TG isolated from subcutaneous depots of growing adult rats plateaued at 85–90% of calculated maximal values at 12 wk ( k = 0.05 day–1), excluding significant dilution by unlabeled α-glycerol phosphate. Turnover of plasma TG, modeled from2H incorporation over 60 min, was 0.06 min–1(half-life 11.5 min). In summary, use of2H2O labeling with MIDA of TG-glycerol allows measurement of new α-glycerol phosphate-derived TG synthesis and turnover. The hypothesis that mesenteric TG is more lipolytically active than other depots, previously difficult to prove by isotope dilution techniques, was confirmed by this label incorporation approach.
VLDL-triglyceride kinetics during hyperglycemia-hyperinsulinemia: effects of sex and obesity
Authors: Mittendorfer Bettina , Patterson Bruce W. , Klein Samuel , Sidossis Labros S.
Published: March 2015

We have previously shown that sex and obesity independently affect basal very low density lipoprotein (VLDL)-triglyceride (TG) kinetics. In the present study, we investigated the effect of hyperglycemia-hyperinsulinemia on VLDL-TG kinetics in lean and obese men and women ( n = 6 in each group). VLDL-TG kinetics were measured during basal, postabsorptive conditions and during glucose infusion (5.5 mg · kg FFM−1· min−1) by using [2H5]glycerol bolus injection in conjunction with compartmental modeling analysis. Basal VLDL-TG secretion in plasma was greater in obese than in lean men (7.8 ± 0.6 and 2.9 ± 0.4 μmol · l plasma−1· min−1; P < 0.001) but was not different in lean and obese women (5.0 ± 1.1 and 5.9 ± 1.1 μmol · l plasma−1· min−1). Glucose infusion decreased the VLDL-TG secretion rate by ∼50% in lean and obese men and in lean women (to 1.5 ± 0.4, 4.0 ± 0.6, and 2.2 ± 0.4 μmol · l plasma−1· min−1, respectively; all P < 0.05) but had no effect on the VLDL-TG secretion rate in obese women (4.9 ± 1.0 μmol · l plasma−1· min−1). These results demonstrate that both sex and adiposity affect the regulation of VLDL-TG metabolism. Glucose and insulin decrease VLDL-TG production in both lean men and lean women; obesity is associated with resistance to the glucose- and insulin-mediated suppression of VLDL-TG secretion in women, but not in men.
Impaired transport of leptin across the blood-brain barrier in obesity is acquired and reversible
Authors: Banks William A. , Farrell Catherine L.
Published: March 2015

Leptin resistance is a major cause of obesity in humans. A major component of this resistance is likely an impaired transport of leptin across the blood-brain barrier (BBB). The fattest subgroup of otherwise normal 12-mo-old CD-1 mice have severely impaired transport of leptin across the BBB. However, it is unknown whether these mice are born with a BBB impairment or acquire it with aging and obesity. Here, we found within an otherwise normal population of CD-1 mice that the 10% fattest mice gained weight throughout a 12-mo-life span, whereas the 10% thinnest mice gained little weight after 3 mo of age. The fattest mice acquired a progressive impairment in their ability to transport leptin across the BBB, whereas the thinnest mice had a rate of transport that did not change with age. Fasting fat mice for 24 h or treating them with leptin resulted in modest weight reduction and development of transport rates for leptin across the BBB similar to those of thin mice. These results show that, in obese CD-1 mice, the impaired transport of leptin across the BBB develops in tandem with obesity and is reversible with even modest weight reduction.
Contribution of plasma proteins to splanchnic and total anabolic utilization of dietary nitrogen in humans
Authors: Fouillet Hélène , Gaudichon Claire , Bos Cécile , Mariotti François , Tomé Daniel
Published: March 2015

Splanchnic tissues are largely involved in the postprandial utilization of dietary amino acids, but little is yet known, particularly in humans, about the relative contributions of different splanchnic protein pools to splanchnic and total postprandial anabolism. Our aim was to develop a compartmental model that could distinguish dietary nitrogen (N) incorporation among splanchnic constitutive, plasma (splanchnic exported), and peripheral proteins after a mixed-protein meal in humans. Eight healthy subjects were fed a single mixed meal containing 15N-labeled soy protein, and dietary N postprandial kinetics were measured in plasma free amino acids, proteins, and urea and urinary urea and ammonia. These experimental data and others previously obtained for dietary N kinetics in ileal effluents under similar experimental conditions were used to develop the compartmental model. Six hours after the mixed-meal ingestion, 31.5, 7.5, and 21% of ingested N were predicted to be incorporated into splanchnic constitutive, splanchnic exported, and peripheral proteins, respectively. The contribution of splanchnic exported proteins to total splanchnic anabolism from dietary N was predicted to be ∼19% and to remain steady throughout the simulation period. Model behavior and its predictions were strongly in line with current knowledge of the system and the scarce, specific data available in the literature. This model provides the first data concerning the anabolism of splanchnic constitutive proteins in the nonsteady postprandial state in humans. By use of only slightly invasive techniques, this model could help to assess how the splanchnic anabolism is modulated under different nutritional or pathophysiological conditions in humans.
Interactive effects of PTH and mechanical stress on nitric oxide and PGE2production by primary mouse osteoblastic cells
Authors: Bakker Astrid D. , Joldersma Manon , Klein-Nulend Jenneke , Burger Elisabeth H.
Published: March 2015

Parathyroid hormone (PTH) and mechanical stress both stimulate bone formation but have opposite effects on bone resorption. PTH increased loading-induced bone formation in a rat model, suggesting that there is an interaction of these stimuli, possibly at the cellular level. To investigate whether PTH can modulate mechanotransduction by bone cells, we examined the effect of 10-9M human PTH-(1-34) on fluid flow-induced prostaglandin E2(PGE2) and nitric oxide (NO) production by primary mouse osteoblastic cells in vitro. Mechanical stress applied by means of a pulsating fluid flow (PFF; 0.6 ± 0.3 Pa at 5 Hz) stimulated both NO and PGE2production twofold. In the absence of stress, PTH also caused a twofold increase in PGE2production, but NO release was not affected and remained low. Simultaneous application of PFF and PTH nullified the stimulating effect of PFF on NO production, whereas PGE2production was again stimulated only twofold. Treatment with PTH alone reduced NO synthase (NOS) enzyme activity to undetectable levels. We speculate that PTH prevents stress-induced NO production via the inhibition of NOS, which will also inhibit the NO-mediated upregulation of PGE2by stress, leaving only the NO-independent PGE2upregulation by PTH. These results suggest that mechanical loading and PTH interact at the level of mechanotransduction.
Effect of intraportal glucagon-like peptide-1 on glucose metabolism in conscious dogs
Authors: Nishizawa Makoto , Moore Mary Courtney , Shiota Masakazu , Gustavson Stephanie M. , Snead Wanda L. , Neal Doss W. , Cherrington Alan D.
Published: March 2015

Arteriovenous difference and tracer ([3-3H]glucose) techniques were used in 42-h-fasted conscious dogs to identify any insulin-like effects of intraportally administered glucagon-like peptide 1-(7–36)amide (GLP-1). Each study consisted of an equilibration, a basal, and three 90-min test periods (P1, P2, and P3) during which somatostatin, intraportal insulin (3-fold basal) and glucagon (basal), and peripheral glucose were infused. Saline was infused intraportally in P1. During P2 and P3, GLP-1 was infused intraportally at 0.9 and 5.1 pmol · kg−1· min−1in eight dogs, at 10 and 20 pmol · kg−1· min−1in seven dogs, and at 0 pmol · kg−1· min−1in eight dogs (control group). Net hepatic glucose uptake was significantly enhanced during GLP-1 infusion at 20 pmol · kg−1· min−1[21.8 vs. 13.4 μmol · kg−1· min−1(control), P < 0.05]. Glucose utilization was significantly increased during infusion at 10 and 20 pmol · kg−1· min−1[87.3 ± 8.3 and 105.3 ± 12.8, respectively, vs. 62.2 ± 5.3 and 74.7 ± 7.4 μmol · kg−1· min−1(control), P < 0.05]. The glucose infusion rate required to maintain hyperglycemia was increased ( P < 0.05) during infusion of GLP-1 at 5.1, 10, and 20 pmol · kg−1· min−1(22, 36, and 32%, respectively, greater than control). Nonhepatic glucose uptake increased significantly during delivery of GLP-1 at 5.1 and 10 pmol · kg−1· min−1(25 and 46% greater than control) and tended ( P = 0.1) to increase during GLP-1 infusion at 20 pmol · kg−1· min−1(24% greater than control). Intraportal infusion of GLP-1 at high physiological and pharmacological rates increased glucose disposal primarily in nonhepatic tissues.
Aberrant insulin-induced GLUT4 translocation predicts glucose intolerance in the offspring of a diabetic mother
Authors: Thamotharan M. , McKnight Robert A. , Thamotharan Shanthie , Kao Doris J. , Devaskar Sherin U.
Published: March 2015

We examined the long-term effect of in utero exposure to streptozotocin-induced maternal diabetes on the progeny that postnatally received either ad libitum access to milk by being fed by control mothers (CM/DP) or were subjected to relative nutrient restriction by being fed by diabetic mothers (DM/DP) compared with the control progeny fed by control mothers (CM/CP). There was increased food intake, glucose intolerance, and obesity in the CM/DP group and diminished food intake, glucose tolerance, and postnatal growth restriction in the DM/DP group, persisting in the adult. These changes were associated with aberrations in hormonal and metabolic profiles and alterations in hypothalamic neuropeptide Y concentrations. By use of subfractionation and Western blot analysis techniques, the CM/DP group demonstrated a higher skeletal muscle sarcolemma-associated ( days 1 and 60) and white adipose tissue plasma membrane-associated ( day 60) GLUT4 in the basal state with a lack of insulin-induced translocation. The DM/DP group demonstrated a partial amelioration of this change observed in the CM/DP group. We conclude that the offspring of a diabetic mother with ad libitum postnatal nutrition demonstrates increased food intake and resistance to insulin-induced translocation of GLUT4 in skeletal muscle and white adipose tissue. This in turn leads to glucose intolerance and obesity at a later stage ( day 180). Postnatal nutrient restriction results in reversal of this adult phenotype, thereby explaining the phenotypic heterogeneity that exists in this population.
Role and regulation of the fibroblast growth factor axis in human thyroid follicular cells
Authors: Cocks Helen C. , Thompson Stuart , Turner Frances E. , Logan Ann , Franklyn Jayne A. , Watkinson John C. , Eggo Margaret C.
Published: March 2015

Thyroidal levels of fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor 1 (FGFR1) are elevated in human thyroid hyperplasia. To understand the significance of this, effects of FGFR1 activation on normal human thyrocyte growth and function in vitro and the regulation of FGF-2 and FGFR1 expression have been examined. FGF-2 stimulated cell growth, as measured by cell counting, and inhibited thyroid function as measured by125I uptake. Sensitivity to FGF-2 disappeared after 7 days, although FGFR1 expression was maintained. Thyroid-stimulating hormone (TSH, 300 mU/l) increased FGFR1 mRNA expression within 4 h and protein expression by 8 h. Exogenous FGF-2 decreased FGFR1 protein. Endogenous FGF-2 levels were low (∼1-2 pg/μg protein), and TSH treatment decreased these by 50%. Protein kinase C (PKC) activation increased FGF-2 mRNA and FGF-2 secretion within 2 h. This effect was enhanced (4.4-fold) when cells were cultured in TSH. We conclude that TSH stimulates FGFR1 but not FGF-2 expression. PKC activation stimulates FGF-2 synthesis and secretion, and TSH synergizes with PKC activators. Increases in FGFR1 or FGF-2 or in both may contribute to goitrogenesis.
Development of models to predict anabolic response to testosterone administration in healthy young men
Authors: Woodhouse Linda J. , Reisz-Porszasz Suzanne , Javanbakht Marjan , Storer Thomas W. , Lee Martin , Zerounian Hrant , Bhasin Shalender
Published: March 2015

Considerable heterogeneity exists in the anabolic response to androgen administration; however, the factors that contribute to variation in an individual's anabolic response to androgens remain unknown. We investigated whether testosterone dose and/or any combination of baseline variables, including concentrations of hormones, age, body composition, muscle function, and morphometry or polymorphisms in androgen receptor could explain the variability in anabolic response to testosterone. Fifty-four young men were treated with a long-acting gonadotropin-releasing hormone (GnRH) agonist and one of five doses (25, 50, 125, 300, or 600 mg/wk) of testosterone enanthate (TE) for 20 wk. Anabolic response was defined as a change in whole body fat-free mass (FFM) by dual-energy X-ray absorptiometry (DEXA), appendicular FFM (by DEXA), and thigh muscle volume (by magnetic resonance imaging) during TE treatment. We used univariate and multivariate analysis to identify the subset of baseline measures that best explained the variability in anabolic response to testosterone supplementation. The three-variable model of TE dose, age, and baseline prostate-specific antigen (PSA) level explained 67% of the variance in change in whole body FFM. Change in appendicular FFM was best explained (64% of the variance) by the linear combination of TE dose, baseline PSA, and leg press strength, whereas TE dose, log of the ratio of luteinizing hormone to testosterone concentration, and age explained 66% of the variation in change in thigh muscle volume. The models were further validated by using Ridge analysis and cross-validation in data subsets. Only the model using testosterone dose, age, and PSA was a consistent predictor of change in FFM in subset analyses. The length of CAG tract was only a weak predictor of change in thigh muscle volume and lean body mass. Hence, the anabolic response of healthy, young men to exogenous testosterone administration can largely be predicted by the testosterone dose.
Differential activation of NF-κB and AP-1 in increased fibronectin synthesis in target organs of diabetic complications
Authors: Chen Shali , Khan Zia Ali , Cukiernik Mark , Chakrabarti Subrata
Published: March 2015

Increased extracellular matrix protein production leading to structural abnormalities is a characteristic feature of chronic diabetic complications. We previously showed that high glucose in endothelial cell culture leads to the upregulation of basement membrane protein fibronectin (FN) via an endothelin (ET)-dependent pathway involving activation of NF-κB and activating protein-1 (AP-1). To delineate the mechanisms of basement membrane thickening, we used an animal model of chronic diabetes and evaluated ET-dependent activation of NF-κB and AP-1 and subsequent upregulation of FN in three target organs of chronic diabetic complications. After 3 mo of diabetes, retina, renal cortex, and myocardium demonstrated increased FN mRNA and increased ET-1 mRNA expression. Increased FN expression was shown to be dependent on ET receptor-mediated signaling, as the increase was prevented by the dual ET receptor antagonist bosentan. NF-κB activation was most pronounced in the retina, followed by kidney and heart. AP-1 activation was also most pronounced in the retina but was similar in both kidney and heart. Bosentan treatment prevented NF-κB activation in the retina and heart and AP-1 activation in the retina and kidney. These data indicate that, although ETs are important in increased FN production due to diabetes, the mechanisms with respect to transcription factor activation may vary depending on the microenvironment of the organ.
A statistical model of diurnal variation in human growth hormone
Authors: Klerman Elizabeth B. , Adler Gail K. , Jin Moonsoo , Maliszewski Anne M. , Brown Emery N.
Published: March 2015

The diurnal pattern of growth hormone (GH) serum levels depends on the frequency and amplitude of GH secretory events, the kinetics of GH infusion into and clearance from the circulation, and the feedback of GH on its secretion. We present a two-dimensional linear differential equation model based on these physiological principles to describe GH diurnal patterns. The model characterizes the onset times of the secretory events, the secretory event amplitudes, as well as the infusion, clearance, and feedback half-lives of GH. We illustrate the model by using maximum likelihood methods to fit it to GH measurements collected in 12 normal, healthy women during 8 h of scheduled sleep and a 16-h circadian constant-routine protocol. We assess the importance of the model components by using parameter standard error estimates and Akaike's Information Criterion. During sleep, both the median infusion and clearance half-life estimates were 13.8 min, and the median number of secretory events was 2. During the constant routine, the median infusion half-life estimate was 12.6 min, the median clearance half-life estimate was 11.7 min, and the median number of secretory events was 5. The infusion and clearance half-life estimates and the number of secretory events are consistent with current published reports. Our model gave an excellent fit to each GH data series. Our analysis paradigm suggests an approach to decomposing GH diurnal patterns that can be used to characterize the physiological properties of this hormone under normal and pathological conditions.
Potassium per kilogram fat-free mass and total body potassium: predictions from sex, age, and anthropometry
Authors: Larsson Ingrid , Lindroos Anna Karin , Peltonen Markku , Sjöström Lars
Published: March 2015

Total body potassium (TBK) is located mainly intracellularly and constitutes an index of fat-free mass (FFM). The aim was to examine whether TBK and the TBK-to-FFM ratio (TBK/FFM) can be estimated from sex, age, weight, and height. A primary study group (164 males, 205 females) and a validation group (161 and 206), aged 37–61 yr, were randomly selected from the general population. TBK was determined by whole body counting, and FFM was obtained by dual-energy X-ray absorptiometry (DEXA; FFMDEXA). The primary study group was used to construct sex-specific equations predicting TBK and TBK/FFM from age, weight, and height. The equations were used to estimate TBK and TBK/FFM in the validation group. The estimates were compared with measured values. TBK in different age ranges was predicted, with errors ranging from 5.0 to 6.8%; errors for TBK/FFM ranged from 2.7 to 4.8%, respectively. By adding FFMDEXAas a fourth predictor, the error of the TBK prediction decreased by approximately two percentage units. In conclusion, TBK and TBK/FFM can be meaningfully estimated from sex, age, weight, and height.
The transcytosis of divalent metal transporter 1 and apo-transferrin during iron uptake in intestinal epithelium
Authors: Ma Yuxiang , Specian Robert D. , Yeh Kwo-Yih , Yeh Mary , Rodriguez-Paris Juan , Glass Jonathan
Published: March 2015

Caco-2 cells grown in bicameral chambers are a model system to study intestinal iron absorption. Caco-2 cells exhibit constitutive transport of iron from the apical (luminal) chamber to the basal (serosal) chamber that is enhanced by apo-transferrin in the basal chamber, with the apo-transferrin undergoing endocytosis to the apical portion of the cell. With the addition of iron to the apical surface, divalent metal transporter 1 (DMT1) on the brush-border membrane (BBM) undergoes endocytosis. These findings suggest that in Caco-2 cells DMT1 and apo-transferrin may cooperate in iron transport through transcytosis. To prove this hypothesis, we determined by confocal microscopy that, after addition of iron to the apical chamber, DMT1 from the BBM and Texas red apo-transferrin from the basal chamber colocalized in a perinuclear compartment. Colocalization was also observed by isolating endosomes from Caco-2 cells after ingestion of ultra-small paramagnetic particles from either the basal or apical chamber. The isolated endosomes contained both transferrin and DMT1 independent of the chamber from which the paramagnetic particles were endocytosed. These findings suggest that iron transport across intestinal epithelia may be mediated by transcytosis.
Atrial natriuretic factor stimulates exocrine pancreatic secretion in the rat through NPR-C receptors
Authors: Sabbatini María E. , Villagra Alberto , Davio Carlos A. , Vatta Marcelo S. , Fernández Belisario E. , Bianciotti Liliana G.
Published: March 2015

Increasing evidence supports the role of atrial natriuretic factor (ANF) in the modulation of gastrointestinal physiology. The effect of ANF on exocrine pancreatic secretion and the possible receptors and pathways involved were studied in vivo. Anesthetized rats were prepared with pancreatic duct cannulation, pyloric ligation, and bile diversion into the duodenum. ANF dose-dependently increased pancreatic secretion of fluid and proteins and enhanced secretin and CCK-evoked response. ANF decreased chloride secretion and increased the pH of the pancreatic juice. Neither cholinergic nor adrenergic blockade affected ANF-stimulated pancreatic secretion. Furthermore, ANF response was not mediated by the release of nitric oxide. ANF-evoked protein secretion was not inhibited by truncal vagotomy, atropine, or Nω-nitro-l-arginine methyl ester administration. The selective natriuretic peptide receptor-C (NPR-C) receptor agonist cANP-(4–23) mimicked ANF response in a dose-dependent fashion. When the intracellular signaling coupled to NPR-C receptors was investigated in isolated pancreatic acini, results showed that ANF did not modify basal or forskolin-evoked cAMP formation, but it dose-dependently enhanced phosphoinositide hydrolysis, which was blocked by the selective PLC inhibitor U-73122. ANF stimulated exocrine pancreatic secretion in the rat, and its effect was not mediated by nitric oxide or parasympathetic or sympathetic activity. Furthermore, CCK and secretin appear not to be involved in ANF response. Present findings support that ANF exerts a stimulatory effect on pancreatic exocrine secretion mediated by NPR-C receptors coupled to the phosphoinositide pathway.
Role of glycogen synthase kinase-3 in TNF-α-induced NF-κB activation and apoptosis in hepatocytes
Authors: Schwabe Robert F. , Brenner David A.
Published: March 2015

Nuclear factor-κB (NF-κB) prevents hepatocytes from undergoing apoptosis during development and liver regeneration. Mice with inactivated glycogen synthase kinase (GSK)-3β die from hepatocyte apoptosis during development due to a defect in NF-κB activation (Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, and Woodgett JR. Nature 406: 86–90, 2000). In this study, we determined the role of GSK-3 in TNF-α-induced NF-κB activation and cell death in primary hepatocytes. LiCl, an established inhibitor of GSK-3, sensitized primary rat hepatocytes toward TNF-α-mediated apoptosis resulting in 90% cell death after 24 h. This was accompanied by increased caspase 8-like and 3-like activities, nuclear fragmentation and DNA laddering. LiCl treatment had no effect on IκB-α degradation, IκB kinase (IKK) activity, NF-κB binding activity, and p65 nuclear import and export, but decreased transcription of the NF-κB-dependent inducible nitric oxide synthase gene and a NF-κB-driven reporter gene. The p65 sequence revealed four potential GSK-3 phosphorylation sites within its COOH-terminal transactivation domains and recombinant GSK-3β phosphorylated glutathione S-transferase (GST)-p65(354–551), but not GST-p65(1–305) in vitro. These results indicate that GSK-3 protects hepatocytes from TNF-α-induced apoptosis through p65 phosphorylation and upregulation of NF-κB transactivation.
Spatial heterogeneity of TNF-α-induced T cell migration to colonic mucosa is mediated by MAdCAM-1 and VCAM-1
Authors: Watanabe Chikako , Miura Soichiro , Hokari Ryota , Teramoto Ken , Ogino Takashi , Komoto Shunsuke , Hara Yuriko , Koseki Seiichiro , Tsuzuki Yoshikazu , Nagata Hiroshi , Granger D. Neil , Ishii Hiromasa
Published: March 2015

Relatively little is known about how recirculation of lymphocytes through the inflamed intestinal mucosa is regulated. The aim of this study was to investigate the dynamic process of T lymphocyte-endothelial cell adhesion in TNF-α-challenged murine colonic mucosa by intravital microscopy. T lymphocytes from spleen (SPL) and intestinal lamina propria (LPL) were fluorescence labeled, and their adhesion to microvessels in the colonic mucosa was observed. In TNF-α (25 μg/kg)-stimulated colonic venules, an enhanced adhesion of SPL and LPL was demonstrated, with dominant recruitment of LPLs. The magnitude of the increased LPL adhesion was more significant in the colon than in the small intestine. These T lymphocyte interactions in the colonic mucosa were significantly reduced by blocking MAbs against either mucosal addressin cell adhesion molecule-1 (MAdCAM-1), VCAM-1, α4-integrin, or β7-integrin but not by anti-ICAM-1. Immunohistochemistry revealed significant MAdCAM-1 expression in the lamina propria and VCAM-1 expression in the submucosa of TNF-α-treated colon. Spatial heterogeneity of MAdCAM-1 and VCAM-1 activation following TNF-α challenge may promote specific T lymphocyte recruitment in the inflamed colonic mucosa.
IV. Genomic engineering for studies of the gastrointestinal tract in mice
Authors: Bullard Daniel C. , Weaver Casey T.
Published: March 2015

Advances in our understanding of the complex, dynamic interactions that exist among the gastrointestinal microflora, the epithelium of the gastrointestinal mucosae, and the immune system have been facilitated by powerful new genetic tools. Recent understanding that the gastrointestinal epithelium performs not only a barrier function but is also an active sensor of the microflora and an important intermediary in regulating and integrating cross-talk between it and cells of the innate and adaptive immune systems provides one of the most fertile and challenging areas for application of these technologies. The intestinal epithelium also represents an important model system for study of programs of cell lineage commitment and differentiation, given its continual and rapid regeneration throughout life and the regional differences in these programs that exist along the gastrocolonic and crypt-villous axes. This review will highlight current and emerging technologies that are available in the mouse model for identification and manipulation of genetic elements that regulate the normal and pathological physiology of the intestinal tissues in the postgenomic era.