bluuebunny/crossref_metadata_2025 huggingface dataset:
The endothelial responses to low‐ and high‐intensity cycling with diesel exhaust exposure (1106.21)
Authors: Giles Luisa , Richard Normand , Ruan Jian , Tebbutt Scott , Carlsten Christopher , Koehle Michael
Published: June 2021

Outdoor exercisers are frequently exposed to air pollution. How the endothelium responds to exercise in air pollution, and how these responses change with exercise intensity is unknown. The purpose of this study was to determine the endothelial responses to low‐ and high‐intensity cycling with diesel exhaust (DE) exposure. Eighteen males aged 24.56.2 yrs performed 30‐minute trials of low (30% of power at VO2peak) and high‐intensity (60% of power at VO2peak) cycling, and rest. For each subject, each trial was performed once breathing filtered air (FA) and once breathing DE (300ug/m3 of PM2.5) for a total of six trials, each separated by 7‐days. Prior to, immediately following, and 1 hour, and 2 hours post‐exposure flow‐mediated dilation (FMD), plasma nitrite/nitrate (NOx), intercellular adhesion molecule (ICAM)‐1, and vascular cell adhesion molecule (VCAM)‐1 were measured. Data were analyzed using repeated‐measures ANOVA. There were no main or interaction effects for FMD. DE exposure did not affect plasma VCAM‐1. Compared to the FA exposure, DE led to lower ICAM‐1 levels following high‐intensity exercise (p<0.05), while plasma NOx was increased (p<0.05). DE exposure does not differentially affect FMD post‐exercise, yet there are significant differences in key biomarkers that warrant further investigation.

Grant Funding Source: Supported by: CASEM, Health Canada, the Fraser Basin Council, NSERC
Prostaglandins contribute in an O2‐dependent manner to exercise hyperaemia following rhythmic and isometric handgrip exercise in young and older healthy subjects (1106.4)
Authors: Junejo Rehan , Ray Clare , Marshall Janice
Published: June 2021

Recreationally active young (Y;18‐25 yrs; n=12) and older (O; 60‐70 yrs; n=7) males performed rhythmic handgrip at 60% maximum voluntary contraction (MVC; 1 sec contraction/relaxation) for 3 min, and 30 min later, isometric handgrip at 60% MVC for 3 min or until exhaustion in a randomized cross over design. They breathed air or 40% O2, and Placebo (P) or cyclooxygenase inhibitor, Aspirin (Asp; 600 mg p.o.). In Y males, forearm vascular conductance (FVC) increased from 0.08±0.01 to 0.59±0.05 conductance units (CU) immediately after isometric contraction during P/air but to only 0.44±0.05*, 0.42±0.07* and 0.42±0.04* CU in P/O2, Asp/air and Asp/O2 respectively (*: P<0.05 vs P/air, Factorial ANOVA). Rhythmic contractions increased FVC from 0.06±0.01 to 0.54±0.08 in P/air and again to only 0.44±0.04*, 0.44±0.04* and 0.45±0.03* CU in P/O2, Asp/air and Asp/O2 respectively. Similarly, in O males, FVC increased from 0.09±0.03 to 0.66±0.09 CU during P/air immediately after isometric contraction and to only 0.50±0.05*, 0.45±0.07* and 0.51±0.05* CU in P/O2, Asp/air and Asp/O2 respectively. Rhythmic contractions increased FVC from 0.08±0.01 to 0.58±0.09 CU in P/air and to only 0.43±0.08*, 0.43±0.07* and 0.49±0.06* CU in P/O2, Asp/air and Asp/O2. Thus we propose that in young and older males, PGs contribute to hyperaemia following rhythmic and isometric contractions and that in both age groups, PG synthesis is O2‐dependent.
Experimental investigation of non‐freezing cold‐induced injury: are young Asian males more susceptible than young white Caucasians and are cyclooxygenase products involved? (1106.5)
Authors: Blevins William , Tipton Michael , Marshall Janice
Published: June 2021

Prolonged or repeated exposure to cold conditions can induce NFCI; Afro‐Caribbeans are more susceptible than white Caucasians. We compared in Caucasian and Asian males (20‐22 years, n=6), responses evoked by 3 immersions of the right leg in water at 15°C for 10 min with 10‐min intervals, the left leg was ~horizontal. In Caucasians, cutaneous red cell flux in right foot (rRCF) fell from 12.8±2.4 to 11.6±1.3, 9.2±1.6* and 8.6±1.5PU in immersions 1, 2 and 3 with some recovery to11.3±2.4, 9.7±2.3* and 9.6±1.6* PU (*: P<0.05 vs baseline). By contrast, in Asians, rRCF fell from 11.1±2.4 to as low as 7.0±0.9*, 6.3±0.8* and 5.8±0.7*PU, with no recovery: 6.8±0.8*, 6.6±0.8*, 6.8±0.7*PU. Caucasians showed no change in contralateral left RCF whereas in Asians, left RCF fell from 25.3±4.2 to 20.1±0.0.9, 20.4±2.8 and 20.5±0.7PU. After COX inhibition (aspirin; 600mg p.o), rRCF fell from 18.0±4.8 to 13.6±4.9*, 11.8±3.7* and 10.9±3.8* during 1st, 2nd and 3rd immersions in Caucasians, with no recovery: 13.5±4.0*, 13.2±4.7* and 11.7±4.0* PU. In Asians, rRCF fell from 7.7±0.9 to 5.9±0.7*, 5.6±0.6* and 5.8±0.6* PU, with no recovery: 5.8±0.9*, 5.6±0.7* and 5.6±0.7*PU. We suggest that young Asians show greater and more persistent local and reflex cutaneous vasoconstrictor responses to cool water immersion than Caucasians and so may be more vulnerable to NFCI; vasodilator COX products may limit cold‐induced vasoconstriction in Caucasians.
Pre‐exercise whole‐body cooling decreases blood volume and oxygenated hemoglobin content in skeletal muscle during submaximal exercise (1106.7)
Authors: Gagnon Dominique , Kyröläinen Heikki , Gagnon Sheila , Herzig Karl‐Heinz , Rintamäki Hannu , Peltonen Juha
Published: June 2021

Dynamic exercise and whole‐body cooling independently increase skeletal muscle tissue deoxygenation, yet their combined effects in muscular circulation are unknown. We investigated the effects of a cold environment on skeletal muscle blood volume and deoxygenation in eleven participants at 50% (walking) or 70% (running) of their peak VO2 for 60 min, in 22°C (NT), and 0°C without (CO) and with (PC) a pre‐exercise whole‐body cooling, which induced an increase in VO2 to 15%VO2peak. Near‐infrared spectroscopy (NIRS) derived signals for hemoglobin changes were measured continuously with NIRS on the vastus lateralis (VL) and the gastrocnemius (GM) muscles to determine changes in oxygenated (O2Hb), deoxygenated (HHb: indicating deoxygenation) and total (tHb: indicating blood volume) myo/hemoglobin concentrations (∆µM.L‐1). Total Hb was lower in PC (‐10.1±5.9 and ‐8.0±7.1 µM.L‐1) compared to NT (‐3.9±3.1 and ‐1.8±4.8 µM.L‐1) and CO (‐3.9±6.6 and 1.9±6.3 µM.L‐1) in walking and running respectively, in the VL but not in GM (p蠄0.001). This change in tHb was matched by O2Hb (p蠄0.001). No differences between conditions were observed in HHb. In conclusion, pre‐exercise whole‐body cooling decreased skeletal muscle blood volume which limited oxygenated hemoglobin availability but deoxygenation of the muscle during submaximal exercise was unaffected by the cold.

Grant Funding Source: Supported by the Jenny and Antti Wihurin Foundation
Chronic carbon monoxide inhalation has a transient effect to reduce weight gain in mice fed a high fat diet (1107.3)
Authors: Hosick Peter , Ahmed Elhaitham , Gousset Monette , Granger Joey , Stec David
Published: June 2021

We recently reported chronic treatment with carbon monoxide (CO) releasing molecules results in prevention of weight gain in mice fed a high fat diet. At present, we sought to determine the effect of chronic CO inhalation on 4 week old lean (Prevent) and 24 week old obese (Reverse) C57BL/6J mice fed a high diet. Two different CO inhalation exposures were used daily for 30 weeks; 28 parts per million (ppm) CO for 2 hours or 200 ppm CO for 1 hour. Body weight, fasted blood glucose and body composition were measured throughout the treatment. Food intake, O2 consumption, CO2 production, motor activity, and body heat production were measured at 28 weeks after the start of the experimental protocol. Compared to high fat diet controls inhalation treatment resulted in prevention and reversal of body weight (Prevent: 36.3gm vs. 42.6gm, p<0.05; Reverse: 45.6gm vs. 52.8gm; p<0.05), fat mass (Prevent: 14.8gm vs. 18.2gm, p<0.05; Reverse: 15.0gm vs. 18.6gm; p<0.05) and, blood glucose (Prevent: 110gm/dl vs. 150gm/dl, p<0.05; Reverse: 124gm/dl vs. 135gm/dl; p<0.05) over the first 10 weeks of treatment, however, this effect was not sustained. No differences were found in any other variables measured. Our results demonstrate that CO inhalation has a transient effect to prevent and reduce body weight which is not sustained in mice fed a high fat diet suggesting chronic CO inhalation therapy is not an effective treatment for long term weight loss.

Grant Funding Source: Supported by grants from the NHLBI, PO1HL‐51971, HL088421 (D.E.S.) and 1T32HL105324 (P.A.H).
Influence of abdominal obesity on circulating oxidative and inflammatory markers in overweight/obese adult men (1107.7)
Authors: Beckstrom Collin , Bammert Tyler , Dow Caitlin , Lincenberg Grace , Diehl Kyle , Greiner Jared , Stauffer Brian , DeSouza Christopher
Published: June 2021

The aim of this study was to determine whether overweight/obese adults with abdominal adiposity exhibit greater oxidative stress and inflammatory burden compared with overweight/obese adults without abdominal obesity. 38 sedentary, adult males free of cardiometabolic disease were studied: 12 normal weight (age 54±2 yr; BMI 22.4±0.6 kg/m2; waist circumference [WC] 82.9+1.4 cm); 15 overweight/obese with WC <102 cm (58+2 yr; 28.1+0.3 kg/m2; 95.5+0.8 cm); and 11 overweight/obese with WC >102 cm (58+3 yr; 29.8+0.7 kg/m2; 107.9+1.7 cm). Plasma concentrations of oxidized low‐density lipoprotein (ox‐LDL), C‐reactive protein (CRP), tumor necrosis factor (TNF)‐α, interleukin (IL)‐6 and IL‐18 were determined by ELISA. Plasma oxLDL (48.7±2.3 U/L), CRP (0.4±0.1 mg/L), TNF‐α (1.5±0.1 pg/mL), IL‐6 (1.3±0.2 pg/mL) and IL‐18 (214±12 pg/mL) levels were lowest in normal weight controls. Importantly, overweight/obese men with abdominal adiposity had significantly higher CRP (2.5±0.7 vs 1.1±0.2 mg/L), IL‐6 (2.9±0.7 vs 1.5±0.3 pg/mL) and IL‐18 (292±15 vs 228±16 pg/mL) levels than overweight/obese adults without abdominal adiposity. There were no group differences in either ox‐LDL (58.4±7.5 vs 55.0±6.0 U/L) or TNF‐α (2.1±0.2 vs 2.6±0.6 pg/mL) amongst the overweight/obese men. These results suggest that abdominal obesity is associated with a more unfavorable inflammatory phenotype in overweight/obese men.
Intrauteruterine growth restriction increases tyrosine hydroxylase expression and propensity for addiction in low birth weight rats (1107.9)
Authors: Ondek Katelynn , Keen‐Rhinehart Erin
Published: June 2021

Obesity is an epidemic of growing concern. Much research has focused on the roles of hypothalamic feeding peptides in energy balance and how these are affected by gestational programming, the impact of the intrauterine environment on adult physiology. Gestational programming may also affect the mesolimbocortical pathway, which projects from the ventral tegmental area (VTA) to the nucleus accumbens and prefrontal cortex in the brain and comprises a general reward system that reinforces natural behaviors such as eating, drinking, and sexual activity. Since low birth weight (LBW) humans tend to exhibit both obesity and a propensity toward addiction, we hypothesized that lack of maternal nutrition may cause baseline changes in dopaminergic activity that lead to perturbations in ingestive behavior. We utilized a traditional rat model of intrauterine growth restriction by placing dams on a 50% food‐restricted diet during the latter half of pregnancy. Offspring had significantly lower birth weights (5.6±0.08g vs. 6.5±0.06g, p<0.01) and significantly higher tyrosine hydroxylase (TH) immunoreactivity in the VTA than controls (159±19 vs. 454±88 A.U., p<0.05), suggesting that they would be more sensitive to rewarding stimuli. Therefore, we examined sucrose consumption in these animals over a 24 hour period and in a limited access paradigm where animals received 10% sucrose for 90 minute intervals. LBW offspring consumed significantly more (29±2ml vs. 23±1ml, p<0.05) when given limited access to sucrose. This suggests that LBW offspring increase ingestive behaviors when given limited access to substances with rewarding properties. Consequently, the dopaminergic reward system deserves closer examination to determine its role in LBW‐related obesity.

Grant Funding Source: Supported by APS UGSRF Fellowship
Mitogen‐inducible gene 6 haploinsufficiency protects mice against streptozotocin‐induced diabetes (1108.11)
Authors: Chen Yi‐Chun , Colvin E. , Fueger Patrick
Published: June 2021

Epidermal growth factor (EGF) and gastrin co‐administration is effective in correcting hyperglycemia and pancreatic beta cell mass deficiencies in various type 1 diabetes (T1D) rodent models, yet the failure in clinical translation of this treatment implies that EGF‐mediated tissue repair is a complicated process warranting further investigation. In this study, we aimed to determine if EGF receptor feedback inhibition by Mitogen‐inducible Gene 6 (Mig6) contributes to the development of T1D. We hypothesized that Mig6 haploinsufficiency, and hence heightened EGFR signaling, would abrogate T1D development in mice. To this end, we treated Mig6 haploinsufficient mice (Mig6+/‐) and their wild‐type littermates (Mig6+/+) with multiple low doses of streptozotocin (STZ) to induce beta cell death and diabetes. We found that whereas STZ‐treated Mig6+/+ mice became diabetic, STZ‐treated Mig6+/‐ mice remained fully glucose tolerant. Concurrently, STZ‐treated Mig6+/‐ mice exhibited preserved levels of circulating insulin following a glucose challenge. As insulin sensitivity was similar between Mig6+/‐ and Mig6+/+ mice, the preserved glucose tolerance in STZ‐treated Mig6+/‐ mice is likely the result of preserved beta cell function. Conversely, Mig6 overexpression in isolated islets compromises glucose‐stimulated insulin secretion. In vitro studies in 832/13 INS‐1 beta cells suggested that Mig6 hinders EGF receptor activation, thereby inhibiting EGF‐mediated DNA damage repair. Finally, STZ‐treated Mig6+/‐ mice have greater beta cell mass recovery at 20 days post‐STZ. These data demonstrate that a reduction in Mig6 promotes beta cell damage repair, thus abating the progression of T1D. Our work suggests that Mig6 may be a novel therapeutic target for T1D.

Grant Funding Source: Supported by NIH grant DK078732 and a DeVault Fellowship from IUSM
GLP‐1 linked to an α2‐adrenergic antagonist enhances glucose disposal (1108.2)
Authors: Stein Lauren , Yosten Gina , Ananthakrishnan Kameswari , Wagner Josef , Lynch Ronald , Samson Willis
Published: June 2021

β‐cells uniquely express both Glucagon Like Peptide‐1 (GLP‐1R) and α2Adrenergic Receptor (α2AR), as a combination. Activation of GLP‐1R potentiates, while α2AR agonists inhibit, glucose stimulated insulin secretion. We hypothesized that GLP‐1 linked to an α2AR antagonist Yohimbine (Yhb) would display enhanced binding and β‐cell specificity, and thus improve glucose disposal compared to GLP‐1 alone. Vehicle, or vehicle containing 1.0 nmole of GLP‐1, Yhb or the GLP‐1/Yhb conjugate were given i.a. 5 min before an i.a. infusion of 1 mg/kg glucose in conscious, unrestrained male rats. Blood was collected for plasma glucose monitoring and insulin determination via a jugular catheter prior to pretreatment, 5 min later prior to glucose infusion, and 1,5,10,15 and 30 min later. GLP‐1 or Yhb alone did not enhance glucose disposal compared to controls. However, glucose levels were cleared significantly faster in rats pretreated with the GLP‐1/Yhb conjugate, returning to baseline already 5 min after glucose infusion. Since, GLP‐1 did not potentiate recovery compared to the GLP‐1/Yhb at the same concentration, these findings are consistent with an increased affinity of GLP‐1, within the bivalent ligand, for binding to β‐cells in vivo. We conclude that the GLP‐1/Yhb conjugate, due to its enhanced β‐cell affinity and specificity, may be a promising therapeutic candidate for control of β‐cell function.

Grant Funding Source: Juvenile Diabetes Research Foundation, NIH HL‐66023
Probing the intra‐islet control of beta cell function (1108.3)
Authors: Elrick Mollisa , Yosten Gina , Salvatori Alison , Samson W.K.
Published: June 2021

Neuronostatin (NST) is produced in pancreatic delta cells from the somatostatin preprohormone, and has been shown to inhibit glucose‐stimulated insulin secretion from beta cells of primary islets, but not from isolated beta cell cultures. We have demonstrated that NST stimulates glucagon release and have hypothesized that this is necessary for the peptide’s in vivo effect to impair glucose tolerance. Here we demonstrate the action of NST to increase proglucagon message in alpha cells and identify a potential mechanism to be via phosphorylation of protein kinase A. The NST receptor, GPR107, is abundantly expressed in the pancreas, in particular in the alpha cell population and both the effect on proglucagon mRNA levels and PKA phosphrylation are absent in alpha cells in which the expression of the NST receptor has been compromised using siRNA. Taken together with our observation that NST infusion in conscious rats impairs glucose tolerance and that plasma levels of the peptide are elevated in the fasted compared to the fed or fasted‐refed state, our studies support the hypothesis that endogenous NST is a physiologically relevant determinant of islet cell function and may contribute to the hyperglucagonemia of the prediabetic state.

Grant Funding Source: NIH HL66023
C‐peptide and insulin at physiological ratios rescue low O2‐induced ATP release from erythrocytes (RBCs) of humans with type 2 diabetes: implications for the prevention of microvascular complications (1108.4)
Authors: Richards Jennifer , Yosten Gina , Kolar Grant , Bowles Elizabeth , Stephenson Alan , Ellsworth Mary , Sprague Randy
Published: June 2021

RBCs participate in the matching of O₂ delivery with tissue need via release of O₂ and ATP when these cells are exposed to low pO₂. This response is deficient in DM2 RBCs. We reported that incubation of healthy human (HH) RBCs with insulin at postprandial levels inhibits low O₂‐induced ATP release. However, in vivo, insulin is co‐released with equimolar amounts of C‐peptide, but due to different degradation rates, C‐peptide levels exceed those of insulin. We reported that co‐incubation of these peptides, at physiological ratios and concentrations, prevents the adverse effects of insulin on ATP release from HH RBCs.

Here we extend these studies to DM2 RBCs. Using Western analysis, we found that the C‐peptide receptor, GPR146, is present on both HH (n=2) and DM2 (n=5) RBCs and pre‐incubation of HH RBCs with an antibody to an external epitope of GPR146 inhibited C‐peptide‐mediated effects (n=3). We next determined that co‐incubation of DM2 RBCs with C‐peptide and insulin at physiological ratios of 1:1 (n=6) or 4:1 (n=8) restored low O₂‐induced ATP release. We also investigated if C‐peptide acts via stimulation of soluble guanylyl cyclase (sGC) activity. The selective sGC inhibitor, ODQ, prevented C‐peptide rescue of low O₂‐induced ATP release in DM2 RBCs. These data suggest a heretofore unrecognized synergism between C‐peptide and insulin that could be important for prevention of the microvascular complications of DM2.

Grant Funding Source: Supported by ADA, AHA and NIH
Effect of vitamin D on insulin resistance and tissue inflammation in western and control diet‐fed mice (1108.7)
Authors: Mutt Shivaprakash , Raza Ghulam , Karhu Toni , Keinänen‐Kiukaanniemi Sirkka , Jarvelin Marjo‐Riitta , Herzig Karl‐Heinz
Published: June 2021

INTRODUCTION: Pandemic Vitamin D (VitD) deficiency is associated with low‐grade chronic inflammation and type 2 diabetes. We hypothesize that vitD could inhibit the inflammatory response and increase the insulin sensitivity.

METHODS: 6‐7 week old, male C57BL/6J mice were fed with ± VitD control (CD; 10% fat) or Western (WD; 41% fat) diets for 15 weeks. The VitD deficient (VDD) control and obese mice were further subdivided into two groups. One group was supplemented with vitD (i.p 50ng/mouse/3x/week), the other with saline for 4 weeks. ITT and GTT performed before and after supplementation. Cytokines and tissue inflammatory gene expressions were analyzed.

RESULTS: In both VDD CD & WD groups 25‐OH‐vitD levels were reduced compared to controls CD & WD (6.3±1.5; 23.5±0.8 vs 93±5.4; 113.1±9.4, P <0.05), and after supplementation levels were significantly increased (73.3±7.8; 62.1±2.6). Both VDD groups and WD mice had an impaired glucose tolerance compared to the CD mice. After VitD supplementation insulin resistance was reduced and glucose tolerance improved. In addition reduced inflammatory cytokines (IL‐1β: 27.2±8.1 vs 167.6±91.9; RANTES: 79.5±13.3 vs 154±69.7) in CD VDD mice but not in WD‐VDD mice.

CONCLUSION: VitD deficiency in both CD and WD fed mice groups induced insulin resistance. Supplementation with VitD in the CD‐VDD improved insulin sensitivity and reduced inflammation, but a similar effect has not been observed in the VDD obese mice.

Grant Funding Source: Supported by Academy of Finland, EVO & Finnish Cultural Foundation North Ostrobothnia Regional funds
Bacterial overload enhances duodenal anion secretion via PKA upregulation (1109.15)
Authors: Akiba Yasutada , Strugatsky David , Kaji Izumi , Kaunitz Jonathan
Published: June 2021

The duodenal mucosa senses luminal bacteria via pattern recognition receptors. Rats, as coprovores, likely populate their foregut with fecal flora, which we hypothesized would enhance anion secretory responses in the duodenum.

We measured short‐circuit current (Isc) in Ussing chambered muscle‐stripped mucosa‐submucosa preparations of rat duodenum. Animals were treated with a suspension of homogenized feces (Fecal, 0.1 g/kg, ig) with or without vehicle (Veh) or omeprazole treatment (OPZ, 10 mg/kg, ig) up to 7 days. Duodenal microflora were assessed by culturing duodenal mucosal scrapes on Brucella blood agar or Lactobacillus‐MRS agar plates.

Basolateral application of forskolin (FK, 10 µM) increased Isc, which was further increased by the addition of carbachol (CCh, 10 µM). The Isc increase in response to FK and CCh was markedly enhanced in OPZ+Fecal group, compared to Veh group, whereas no differences were noted among Veh+Fecal, OPZ, or Veh groups. Acetazolamide‐sensitive Isc was increased in OPZ+Fecal group, consistent with enhanced HCO3‐ secretion. Expression of protein kinase A (PKA) catalytic domain a and b, and regulatory domain 1a and 2a was significantly increased in Veh+Fecal and OPZ+Fecal groups, while the expression of the cystic fibrosis transmembrane conductance regulator (CFTR), cyclooxygenase (COX)‐2 or tumor necrosis factor (TNF)‐α was not changed. OPZ treatment increased the detection frequency of lactobacillus in the duodenum, and OPZ+Fecal treatment markedly increased lactobacillus colony counts.

These results suggest that increased foregut bacterial exposure enhances the anion secretory response to FK in the duodenal mucosa. Since lactobacillus is a major probiotics, upregulation of PKA by probiotics may be a novel pathway to modulate the duodenal mucosal defenses.

Grant Funding Source: Supported by VA Merit Review, NIH R01 DK54221
Estrogen, progesterone and prolactin are involved in the sex‐dimorphic regulation of the renal NaCl cotransporter (1109.3)
Authors: Rojas‐Vega Lorena , Reyes‐Castro Luis , Meade Patricia , De los Heros Paola , Bautista Rocio , Hadchouel Juliette , Zambrano Elena , Gamba Gerardo
Published: June 2021

Pregnancy, lactation and the female sexual cycle are physiological conditions in which volume retention is required. It has been shown that the renal thiazide‐sensitive NaCl cotransporter (NCC) expression is higher in female than in male rats, but little is known about the mechanism. In this study we observed that, in contrast to male rats, female rats exhibit a higher response to a single ip injection of a thiazide type diuretic, but not to furosemide or amiloride. In the kidney, female rat and mice exhibit higher expression and T‐58 phosphorylation of NCC than males, as well as higher expression and S‐383 phosphorylation of the kinase SPAK. The difference disappeared after ovariectomy. No effect of orquiectomy was observed. The administration of β‐estradiol or progesterone in ovariectomized rats restored the increased phosphorylation of NCC. A role for prolactin in NCC regulation, however, cannot be ruled out because estrogens enhance prolactin secretion. In addition, prolactin is a known osmoregulator in teleost fish. We thus assessed the expression and phosphorylation of NCC in mice lacking the prolactin receptor (PRLR‐/‐). The difference between female and male persisted in PRLR‐/‐ mice, but this could be due to ovarian hormones. However, in male PRLR‐/‐ mice phosphorylation of NCC was dramatically reduced, when compared to PRLR+/+ littermates, suggesting that by lacking estrogen and progesterone, male PRLR‐/‐ mice are more suitable model to document the NCC regulation by prolactin. Supporting this hypothesis, ex vivo male rat kidneys perfusion with prolactin demonstrated a dramatic increase in NCC phosphorylation when compared with vehicle‐perfused controls. We thus conclude that estrogen, progesterone, and prolactin are positive activators of NCC, which in turn play a role in the volume retention observed in female physiological conditions such as the female sexual cycle, pregnancy and lactation.
Intestinal epithelial barrier dysfunction protects against pathogen translocation during initial infection (1111.2)
Authors: Edelblum Karen , Singh Gurminder , El Bissati Kamal , McLeod Rima , McCormick Beth , Turner Jerrold
Published: June 2021

Myosin light chain kinase (MLCK) is essential for pathophysiological epithelial tight junction regulation. Transgenic mice with intestinal epithelial‐specific expression of constitutively‐active MLCK (CA‐MLCK) demonstrate increased permeability and susceptibility to immune‐mediated colitis. However, the effect of barrier dysfunction on enteric infection remains unknown. Within 30 min after mucosal application of DsRed‐labeled Salmonella typhimurium (SL3201 108 CFU), CA‐MLCK mice exhibited a 5‐fold reduction in bacterial translocation compared to wildtype (WT). This protection also occurred 1 h after oral infection with Toxoplasma gondii, in which tachyzoite invasion was reduced in CA‐MLCK mice relative to WT, 1.5±0.6 vs 4.0±1.0 tachyzoite/mm2 villus, respectively. Intraepithelial lymphocytes expressing the γδ T‐cell receptor (γδ IEL) protect against pathogen invasion. Time‐lapse confocal microscopy revealed enhanced γδ IEL surveillance of the villous epithelium in CA‐MLCK mice compared to WT with more rapid migration (4.2±0.1 vs 3.8±0.1μm/min) and reduced localization to the lateral intercellular space (26±1.7 vs 32±3.3%). However, loss of γδ T cells in CA‐MLCK mice did not alter Salmonella transmigration compared to WT. These data indicate that increased γδ IEL epithelial surveillance is not sufficient for the reduced pathogen invasion observed during early infection in CA‐MLCK mice.

Grant Funding Source: Supported by NIH DK61931 and DK68271, UChicago DDRCC, Gastro‐intestinal Research Foundation
Stabilisation of epithelial hypoxia‐inducible factor reverses colitis through accelerated restitution (1111.3)
Authors: Goggins Bridie , Minahan Kyra , Kostakis Anna , Shalwitz Robert , Horvat Jay , Keely Simon
Published: June 2021

During mucosal inflammation, changes in metabolic activity and vascular tissue damage, lead to a reduction in tissue‐oxygen tension (hypoxia), in which healing processes such as re‐epithelialisation occur. Previous work has shown that pharmacological induction of hypoxia‐inducible factor (HIF), a global transcriptional regulator of the hypoxic response, by prolyl hydroxylase inhibitors (PHDi) is protective in murine models of colitis.

We have previously identified induction of integrin β1 (ITGB1) during hypoxia. As integrins play critical roles in epithelial wound healing, we hypothesised that pharmacological HIF activation accelerates epithelial restitution through integrin‐dependent pathways. We aimed to investigate the relative induction of integrin β1 heterodimers in epithelial cells by PHDi in vivo and in vitro.

Colitis was induced in BalbC mice by rectal instillation of 2.5% TNBS. PHDi (Akebia‐4924, [5mg/Kg]) was administered by oral gavage. Colitis was monitored endoscopically over 7 Days with the Karl Storz coloview endoscope. Biopsies were taken on alternate days with a 20‐french biopsy forceps. Mice were euthanised and tissues and colonic epithelial cells were harvested for mRNA and protein. Scratch assays were performed on Caco‐2 monolayers seeded on 6 well plates.

TNBS colitis led to a significant increase in ITGB1 transcript in epithelium, correlating (R2=0.71) disease severity. Mice treated with PHDi demonstrated advanced endoscopic healing compared to vehicle treated animals, concurrent with an earlier induction of ITGB1 and induction of epithelial integrins α3 and α6. Caco‐2 monolayers show accelerated wound healing in scratch assays when treated with 10mM PHDi which was inhibited by antibodies functionally targeting α3β1 and α6β1 integrin homodimers. These data suggest that mucosal HIF activation promotes healing through accelerated induction of epithelial intregrins.

Grant Funding Source: National Health and Medical Research Council
Risk factors of diseases gastrointestinal path (1112.1)
Authors: Imashev M. , Dubitsky A. , Fursov A.
Published: June 2021

Determination of risk factors of diseases of a gastrointestinal path at teenagers.

Material. Under supervision there were 224 teenagers from 15 to 18 years, from them 112 teenagers with pathology of a digestive tract and 112 control group. Diagnostics was carried out on the basis of standard criteria.

Results. Non‐compliance with a diet took place at 33 (29,5%) teenagers of the main group, at 11 (9,8%) control (3,0 points). The frequent use of hot and smoked dishes was observed at 17,8% teenagers of the main group and at 8% control (2,0 points). Negative emotions are established almost at each 5‐6 teenagers ‐ 17,8% the main group and at 8% control. Bad living conditions are established at 24,1% 1 groups and at 13,4% 2 groups 1,8 points. In 1 group have addictions more than 28%, from them smoke 22,3%, take alcoholic beverages 8,9%. In control group 7,1% and 2,7%, respectively (3,0 and 3,3 points).

1 group (low risk) included teenagers at whom total amount of points on risk factors didn't exceed 5; the 2nd group included teenagers with a score from 5 to 10; the 3rd group ‐ high risk ‐ included children with the sum of the gained points 10,0 and more. Within a year medical and improving events at 35 teenagers of group of high risk were held. In group of high risk there were only 19 teenagers as they passed to groups of an average and low risk, and the part of teenagers was struck off the register. The indicator efficiency of improvement made 45,7%.
Ghrelin and des‐acyl ghrelin stimulate glucose output in mouse primary hepatocytes (1114.2)
Authors: Callaghan Brid , Hunne Billie , Montanholi Martins Vitor , Brock James , Furness John
Published: June 2021

In humans, i.v. administration of ghrelin elicits an increase in glucose levels. In pig, ghrelin stimulates whereas des‐acyl ghrelin inhibits glucose output from hepatocytes. The aims of the current study were to pharmacolgically characterise the receptor responsible for the changes in glucose release from mouse hepatocytes in response to ghrelin family ligands and compare this profile to the profile of the known ghrelin receptor expressed in HEK293 cells. Ghrelin activated GHSR1a in transfected HEK293 cells causing a release of intracellular calcium with an EC50 of 2.5 nM. Des acyl ghrelin had an EC50 > 2 µM. Hepatocytes were cultured overnight and incubated with 100 nM ghrelin, 100 nM des‐acyl ghrelin, 1 µM glucagon or vehicle for 20 minutes. Ghrelin, des‐acyl ghrelin and glucagon all significantly increased glucose output above basal levels. When glycogen stores were depleted no significant changes were seen in glucose output in response to ghrelin and des acyl ghrelin. In mouse hepatocytes ghrelin and des acyl ghrelin both stimulate glucose release. The released glucose is a product of glycogenolysis. It is likely that the actions of ghrelin and des acyl ghrelin in mouse hepatocytes are via a novel receptor as ghrelin and des acyl ghrelin are equipotent at stimulating glucose release whereas at GHSR1a des acyl ghrelin is 1000 fold less potent than ghrelin, and GHSR1a is absent or barely detectable in liver.
Nitric oxide synthase inhibition exacerbates liver injury in an obese rat model of NAFLD (1115.3)
Authors: Sheldon Ryan , Padilla Jaume , Jenkins Nathan , Laughlin M. , Rector R.
Published: June 2021

We tested the hypothesis that chronic disruption of constitutive nitric oxide (NO) production via the non‐selective NO synthase (NOS) inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME) would intensify liver injury in a rat model of obesity, insulin resistance, and nonalcoholic fatty liver disease (NAFLD). Obese Otsuka Long‐Evans Tokushima Fatty (OLETF) and lean Long‐Evans Tokushima Otsuka (LETO) rats (16‐wks) received either control or L‐NAME (65‐70 mg/kg/d) containing drinking water (n=10/group) for 4‐wks. L‐NAME treated OLETF and LETO rats had significantly (p < 0.05) reduced serum NO metabolites (NOX) and food intake. Despite reducing % body fat (‐2.5%; p < 0.05) in OLETF rats, L‐NAME treatment increased hepatic triacylglycerol (TAG) content (+40%; p < 0.05) compared to control OLETF rats. In addition, L‐NAME treatment caused a shift toward M1 macrophage polarity as indicated by elevated CD11c (p<0.05) and IL‐1β (p=0.07) mRNA in the OLETF rats. Markers of total and resident macrophages (CD68 and CD163 mRNA, respectively), fibrosis (TGFβ mRNA), and cell adhesion (MCP‐1 and ICAM mRNA) were unaffected by L‐NAME treatment in both groups. In conclusion, chronic NOS inhibition appears to aggravate liver injury by increasing expression of macrophage activation‐related genes associated with insulin resistance and by accelerating hepatic TAG accumulation in obese, insulin resistant rats.

Grant Funding Source: Supported by a Life Sciences Fellowship (RDS), NIH HL‐36088 (MHL), and VHA‐CDA2 BX001299‐02 (RSR)
Secretory and absorptive dysfunctions in ob/ob mouse jejunum are sex‐dependent (1117.1)
Authors: Al‐Nakkash Layla , Leung Lana
Published: June 2021

Sex‐dependent differences in intestinal absorption and intestinal secretion in the ob/ob leptin‐deficient obese mouse are poorly understood. We used male and female ob/ob and lean mice aged ~14 weeks. The goal of this study was to characterize jejunal function in this clinically relevant mouse model. We measured transepithelial short circuit current (Isc), a measure of chloride secretion, across freshly isolated segments of jejunum from ob/ob and lean mice. Compared to lean controls, basal Isc was significantly decreased by 24 µA/cm2 (P<0.05, n=9) in ob/ob females and significantly decreased by 30 µA/cm2 (P<0.05, n=10‐11) in ob/ob males. Reduced basal Isc in ob/ob males is partly attributed to a significant 2‐fold decrease in CFTR expression. The contribution of key transporters involved in promoting chloride secretion are currently under evaluation. While crypt depth was unchanged in ob/ob males, the number of goblet cells/crypt was 2‐fold greater and this may also account for, in part, the reduced basal Isc in ob/ob males. Villi length and total small intestinal length were both significantly increased in ob/ob female and male mice, suggesting a greater surface area for absorptive function. These structural changes likely contribute towards the obese phenotype. Expression of the fructose transporter protein, GLUT5 (normalized to GAPDH), was significantly increased 1.9‐fold (P<0.05, n=12‐15) in male ob/ob mice compared to leans, which likely contributes to the diabetic/obese phenotype of ob/ob males. Expression of the glucose co‐transporter protein, SGLT1 (normalized to GAPDH), was significantly decreased 30% (P<0.05, n=8‐9) in female ob/ob mice compared to leans. These data suggest that basal jejunal Isc in ob/ob mice is deficient, and may contribute towards slower transit times in the gastrointestinal tract in the ob/ob mice. Interestingly, while both female and male ob/ob mice are obese, the specific secretory and absorptive dysfunctions are not comparable in both sexes.

Grant Funding Source: Supported by NIH R15 DK071625‐01A2
Sucralose causes a concentration dependent metabolic inhibition of the gut flora Bacteroides, B. fragilis and B. uniformis not observed in the Firmicutes, E. faecalis and C. sordellii (1118.1)
Authors: Rettig Scott , Tenewitz Jake , Ahearn Greg , Coughlin Charles
Published: June 2021

The gut microbiota is composed mainly of members from the phyla Bacteroides and Firmicutes. Others have shown a correlation of obesity with a reduction in the Bacteroides’ ability to grow normally and maintain their role in the gut. Sucralose is a ‘non‐metabolizable’ chlorinated sucrose derivative and the synthesized ingredient in the artificial sweetener Splenda®. Turbidity data obtained from active cultures showed a differential effect of sucralose on the growth curves between members of these Phyla. Sucralose had little effect on two Firmicutes, E. faecalis and C. sordellii, while there was a concentration dependent inhibition of growth of Bacteroides, B. fragilis and B. uniformis. Preliminary results of sucrase enzyme assays may demonstrate considerable competitive inhibition in the presence of sucralose. Furthermore, preliminary transport tests may have displayed differential results between the two phyla suggesting two putative means of metabolic inhibition that explain the reported growth curves. It must be considered that sucralose has the ability to alter gut flora composition by these differential metabolic findings and the negative health impacts from this imbalance must be addressed.
Cultured milk bacterial isolates survive well in digestive tract conditions and could be potential probiotic candidates (1118.4)
Authors: Boyiri B , Kotala J , Lampson B , Onyango E
Published: June 2021

Probiotics are viable non‐pathogenic microbes that positively affect host health. Our goal is to understand the influence of probiotics on the intestinal barrier function. In this study bacterial isolates from traditional cultured milk from Kenya were screened for their ability to withstand digestive tract conditions and thence their potential as probiotic candidates. Bacteria in the sample were isolated and identified by single colony PCR sequence match analysis of the 16s rRNA gene. Identified isolates included Lactobacillus spp (6 isolates), Acetobacter spp (2 isolates), Bacillus spp (2 isolates) and 1 Staphylococcus sp. Isolates were examined for acid stability over a 3‐h period, acid and/or bile stability over a 9‐h period, antimicrobial activity, and antibiotic sensitivity to ampicillin, bacitracin, chloramphenicol, erythromycin, kanamycin, penicillin, streptomycin and tetracycline. Two Lactobacillus spp isolates were stable in acid and/or bile, showed antimicrobial activity against K. pneumonia and P. aeruginosa and were sensitive to erythromycin, kanamycin, streptomycin and tetracycline. The remaining 4 Lactobacillus isolates were unstable in acid and/or bile, showed antimicrobial activity against K. pneumonia and P. aeruginosa and were sensitive to all tested antibiotics. The Bacillus spp isolates were acid and/or bile stable, showed no antimicrobial activity, and were susceptible to all the tested antibiotics. The Staphylococcus sp and 2 Acetobacter spp were acid and bile unstable and did not have antimicrobial activity and were sensitive to the antibiotics tested. In conclusion, the Lactobacillus isolates that showed stability in acid/bile conditions, demonstrated antimicrobial activity and were sensitive to antibiotics could be potential probiotic candidates.

Grant Funding Source: East Tennessee State University Research Development Committee Small Grants Program
CagA+ Helicobacter pylori induces proliferation and expression of Sonic hedgehog in fundic organoid model (1119.4)
Authors: Schumacher Michael , Aihara Eitaro , Feng Rui , Zavros Yana
Published: June 2021

Helicobacter pylori (H. pylori) infection leads to aberrant cell proliferation and dysregulated Hedgehog signaling in the stomach. The organoid model is an emerging in vitro system whereby isolated gastric glands are cultured into three‐dimensional spheres that express cells present in the original tissue. This model allows for the study of epithelial responses to infection in a system independent of recruited inflammatory cells. We hypothesize that H. pylori induces cell proliferation and acute expression of Sonic hedgehog (Shh) in the fundic organoid model in the absence of recruited circulating host factors. Fundic organoids were generated from dissociated gastric glands of control or transgenic mice lacking Shh expression in the parietal cell (PC‐ShhKO). Expression of mature cell lineage markers was confirmed by RT‐PCR and immunofluorescence staining. Colonization and epithelial attachment was confirmed by silver stain, live‐imaging and quantitative re‐culture. Organoids injected with cagA+ but not cagA‐deficient H. pylori had significantly increased cell proliferation as measured by EdU staining. Proliferation was associated with c‐Met phosphorylation as determined by western blot. H. pylori induced Shh at 1 day post‐injection in control organoids but not those generated from PC‐ShhKO mice, and this effect was inhibited by NFκB inhibition. Inhibition of CXCL1/2 signaling, a putative mediator of Shh induction, did not block H. pylori‐induced Shh expression. This report demonstrates a uniquely beneficial in vitro approach to study the host response to H. pylori interactions with the gastric epithelium, independent of recruited hematopoietic factors. Fundic organoids recapitulate the in vivo responses to H. pylori of cell proliferation and Shh induction and provide mechanistic insight demonstrating 1) cagA dependence and c‐Met phosphorylation association with H. pylori‐induced proliferation and 2) NFκB requirement for H. pylori‐induced gastric Shh expression.

Grant Funding Source: Supported by NIH 1R01DK083402‐01 (Y. Zavros) and AGA: Funderburg Research Award (Y. Zavros)
Improving post‐traumatic stress disorder biomarkers through matching patients and controls on multiple criteria (1120.10)
Authors: Donohue Duncan , Miller Stacy‐Ann , Gupta Ellen , Yang Ruoting , Guatam Aarti , Hammamieh Rasha , Jett‐Tilton Marti
Published: June 2021

Molecular typing of psychological disease depends on a variety of complicated environmental and phenotypic factors. The quality of results critically depends on the cohort selection criteria, including those criteria used to match case and controls. Our PTSD biomarker study initially matched patient groups on age and self‐reported race. Further analysis has shown striking differences in the types and amounts of medications used by PTSD patients vs. controls. Here, we present an approach to simultaneously match patient groups across age, ancestry, and medication use applying random sampling methods. We compare receiver operating characteristics to quantify the degree to which matching on these three variables generates blood mRNA expression biomarkers which better discriminate patients from controls. Work is in progress to fine‐tune matching by incorporating the relative effect size of these three measures.

Disclaimer: Research was conducted in compliance with the Animal Welfare Act and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army. Human blood sample use protocol was conducted at USACEHR under HRPO Log number A‐15459.f.
Base‐resolution maps of 5‐methylcytosine and 5‐hydroxymethylcytosine in Dahl salt‐sensitive rats (1120.8)
Authors: Liu Yong , Liu Pengyuan , Yang Chun , Cowley Allen , Liang Mingyu
Published: June 2021

Analysis of 5‐hydroxymethylcytosine (5hmC) at single‐base resolution has been largely limited to studies of stem cells or developmental stages. Given the potential importance of epigenetic events in hypertension, we have analyzed 5hmC and 5‐methylcytosine (5mC) at single‐base resolution in the renal outer medulla of the Dahl salt‐sensitive SS rat and examined the effect of disease‐relevant genetic or environmental alterations on 5hmC and 5mC patterns. Of CpG sites that fell within CpG islands, 11% and 1% contained significant 5mC and 5hmC, respectively. 5mC levels were substantially higher for genes with lower mRNA abundance and showed a prominent nadir around the transcription start site. In contrast, 5hmC levels were lower in genes with lower expression. Substitution of a 12.9 Mbp region of chromosome 13, which attenuates the hypertensive and renal injury phenotypes in SS rats, or exposure to a high‐salt diet, which induces the disease phenotypes, was associated with differential 5mC or 5hmC in several hundred CpG islands. Nearly 80% of the CpG islands that were differentially methylated in response to salt and associated with differential mRNA abundance were intragenic CpG islands. The substituted genomic segment had significant cis effects on mRNA abundance but not DNA methylation. The study established base resolution maps of 5mC and 5hmC in an in vivo model of disease and revealed several characteristics important for understanding the contribution of 5mC and 5hmC to hypertension and other complex diseases.

Grant Funding Source: Supported by the NIH.
Ectoderm specific deletion of Jak2 results in developmental and neuronal abnormalities (1121.4)
Authors: Park Sung , Wagner Kay‐Uwe , Sayeski Peter
Published: June 2021

Janus kinase 2 (Jak2) is a non‐receptor tyrosine kinase that is expressed in a number of mammalian tissues. Within the central nervous system, Jak2 has been associated with neuroproliferation, glial differentiation, synaptic plasticity, energy homeostasis, brain tumors, and neurodegenerative disorders. Recent clinical studies that have used Jak2 inhibitor therapy for the treatment of hematological disorders have noted an adverse event in the form of neuropathies in various ectoderm derived sensory organs as well as confusional states of mind. However, the precise physiological/patho‐physiological roles that Jak2 plays in ectodermal derived organs is not clear. Here, we hypothesized that Jak2 plays a critical role in the development of the ectoderm in mice. To test this, we created a tissue specific knockout mouse that lacks Jak2 in ectodermal derived organs. When compared to wild type littermate controls, these mice have a number of abnormalities in their reproductive organs, eyes, and digits. In addition, the mice had a marked alopecia and a behavioral disorder characterized by aimless and repeated circling within the cage. Interestingly, the penetrance of phenotypes was observed in 30% ‐ 50% of the homozygous mutant mice, thereby suggesting a role for other modifiers in these abnormalities. Collectively, these results indicate that Jak2 plays a critical role in the development of the mammalian ectoderm. Furthermore, because of the current use of Jak2 inhibitors in humans, these mice can serve as a valuable research tool for investigating neurotoxicity that is secondary to the loss of Jak2 function in ectoderm tissues.

Grant Funding Source: Supported by NIH and AHA
NHE8 in human conjunctival epithelial cells: expression and regulation (1122.3)
Authors: Zhao Yang , Li Jing , Wang Mingwu , Ghishan Fayez , Xu Hua
Published: June 2021

Dry eye disease (DED) is a disorder of the tear film by symptoms of dryness and irritation. Although the pathogenesis of dry eye is not fully understood, it is recognized that inflammation plays prominent role in the development and propagation of the debilitating condition. We have showed previously that loss of NHE8 function in mice resulted in dry eye syndrome. Therefore, we wanted to know if inflammatory cytokines (IL‐1β, IL‐6, IL‐17A and TNF‐α) which are detected in dry eye patients regulate NHE8 expression in the conjunctival tissue. Our results showed that NHE8 protein abundance was dramatically reduced in cells exposured to IL‐6, IL‐17A and TNF‐α in human conjunctival epithelial cells. The reduction on NHE8 protein expression was likely through inhibition of NHE8 mRNA expression in the case of IL‐1β, IL‐17A and TNF‐a. In summary, our study suggested that inflammatory cytokines regulate NHE8 expression at both protein and mRNA levels in dry eye condition.

Grant Funding Source: Supported by NIH grant R01DK073638