bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of hyperoxia on contraction‐induced forearm blood flow dynamics in young healthy adults (1106.13)
Authors: Ranadive Sushant , Joyner Michael , Walker Branton , Taylor Jennifer , Casey Darren
Published: June 2021

Hyperoxia can cause substantial reductions in peripheral and coronary blood flow at rest and during exercise. We tested the hypothesis that hyperoxia may increase the time for forearm blood flow (FBF) to reach steady state (SS) levels during exercise. Young (n=11; 28±2yr) healthy adults performed rhythmic forearm contractions at 10% of their maximum voluntary contraction for 5 minutes. Each duty cycle consisted of 1 second contraction and 2 second relaxation. Brachial artery diameters and velocities were measured using Doppler Ultrasound and forearm blood flow (ml/min) was calculated from the mean velocity and diameter. The dynamics of FBF at the onset of exercise were assessed by calculating the slope of the FBF response for every duty cycle between baseline and steady state, and the number of duty cycles to reach steady state. The slope of the line plotted from baseline FBF to SS, had a significantly attenuated slope under hyperoxia (5.4±1.1 ml/min/cycle) as compared to normoxia (10.6±2.6 ml/min/cycle). In addition, the number of duty cycles performed under hyperoxia (33±4 cycles) was significantly higher as compared to normoxia (25±4 cycles) (P<0.02). Our data suggest that hyperoxia attenuates the contraction‐induced FBF from baseline to SS as compared to normoxia.
Involvement of NAD(P)H oxidase in acrolein‐induced oxidative stress (1106.14)
Authors: Yousefipour Zivar , Newaz Mohammad
Published: June 2021

Acrolein toxicity has been linked to oxidative stress & inflammation. NAD(P)H oxidase plays an important role in oxidative stress. gp91phox, an essential component of NAD(P)H oxidase, maintains the functionality of the enzyme. The precise contribution of NAD(P)H in acrolein‐induced toxicity is not fully explored. We investigated the involvement of NAD(P)H oxidase in acrolein toxicity using gp91phox knock‐out (KO) mice. Male gp91 KO mice (20‐25 g) or wild type (WT) control were treated with acrolein (0.5 μg/kg; 1 week). Animals were sacrificed and liver was used for biochemical analysis. KO mice generated low (1.43±.02 pg/μg protein) free radical as evident by 8‐Isoprostane compare to the WT mice (2.19±0.1). Acrolein increased 8‐Isoprostane in WT (P<0.05) but not in KO mice. Xanthine Oxidase (XO) activity was higher (p<0.05) in KO (0.56±0.06 μunit/μg protein). Acrolein reduced XO in KO mice while significantly increased it in WT. Cycloxygenase (COX) activity was not different between WT and KO mice although acrolein increased COX in WT. KO mice exhibited a significantly low (2.1±0.2 μmol/mg protein) total antioxidant status (TAS) compared to the WT (3.5±0.3). Acrolein reduced TAS in both WT and KO mice in an equal manner. These data suggests that NAD(P)H oxidase contributes significantly in acrolein‐induced oxidative stress. We suggests that in absence of NAD(P)H oxidase XO and COX play a definitive role in this process. We also suggest that besides generating free radical, acrolein reduces antioxidant ability thus compounding the effects of oxidative stress.

Grant Funding Source: Supported by NIH‐SC3
Tempol improves cutaneous thermal hyperemia through increasing nitric oxide bioavailability in young smokers (1106.2)
Authors: Fujii Naoto , Brunt Vienna , Francisco Michael , Minson Christopher
Published: June 2021

We recently found that young cigarette smokers display cutaneous vascular dysfunction relative to non‐smokers, which is partially due to reduced nitric oxide (NO) synthase (NOS)‐dependent vasodilation. In this study, we tested the hypothesis that reducing oxidative stress improves NO bioavailability, enhancing cutaneous vascular function in young smokers. In ten healthy young male smokers, cutaneous vascular conductance (CVC) during local heating to 42ºC at a rate of 0.1ºC/sec was evaluated as laser‐Doppler flux divided by mean arterial blood pressure. We evaluated plateau CVC during local heating, which is known to be highly dependent on NO, at four intradermal microdialysis sites: 1) Ringer’s (control), 2) 10μM 4‐hydroxy‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl (Tempol), a superoxide dismutase mimetic, 3) 10mM Nω‐Nitro‐L‐arginine (L‐NNA), a non‐specific NOS inhibitor, and 4) a combination of 10μM Tempol and 10mM L‐NNA. Tempol increased the plateau CVC compared with the Ringer’s site (90.0±2.3 vs. 77.6±3.9%max, P=0.049). Plateau CVC at the combination site (56.8±4.5%max) was lower than the Ringer’s site P=0.002), and was not different from the L‐NNA site (55.1±4.6%max) (P=0.778), indicating the Tempol effect was exclusively NO‐dependent.

These data suggest that in young smokers, reducing oxidative stress improves cutaneous thermal hyperemia to local heating by enhancing NO production.

Grant Funding Source: This study is supported by NIH HL081671 and The Uehara Memorial Foundation
Sex differences observed in forearm exercise vasodilation are not mediated through differences in nitric oxide or prostaglandin signaling (1106.3)
Authors: Kellawan Jeremy , Johansson Rebecca , Harrell John , Trierweiler Joshua , Crain Meghan , Sebranek Joshua , Walker Benjamin , Eldridge Marlowe , Schrage William
Published: June 2021

We hypothesized exercise vasodilation in skeletal muscle differs between sexes due to nitric oxide synthase (NOS) and cyclooxygenase (COX) signalling. 39 healthy adults (19 females, F, 20 males, M, 26 ± 1 yrs) completed two 10‐min bouts of dynamic forearm exercise at 15% effort, separated by 20 min rest. After 5 min of control exercise, NOS or COX was inhibited during the final 5 min of exercise by intra‐arterial infusion of L‐NMMA or ketorolac, respectively. During min 5‐10 of the second exercise bout the remaining drug was infused to achieve double blockade (DB). Forearm blood flow (FBF; echo and Doppler ultrasound), arterial pressure (brachial catheter), and forearm lean mass (DEXA) were measured to calculate relative forearm vascular conductance (FVC) = FBF/100mmHg/100g lean mass. Results are mean ± SE. Females exhibited greater vasodilation than Males in control exercise (Δ FVC, 19±1 vs. 14±1, P < 0.01). L‐NMMA reduced FVC similarly (F: Δ ‐2.3 ± 1.3 vs. M: Δ – 3.7 ± 0.8, p = 0.85). In contrast, ketorolac increased FVC similarly in Females and Males (F: Δ 2.1 ± 1.3 vs. M: Δ 2.2± 1.9, p = 0.83). Despite Females exhibiting a greater exercise FVC, DB caused similar Δ FVC between the sexes (0.89). Females have a greater exercise vasodilatory response. NOS, COX, and DB responses are similar, thus, exercise vasodilation differences between sexes are due to a NOS‐COX independent mechanism(s).

Grant Funding Source: Supported by NIH 144PRJ52FK
Cholecystokinin activates a Ca2+‐dependent Cl‐ conductance in intestinal nodose neurons that is inhibited in diet‐induced obesity and may suppress satiety (1107.8)
Authors: Wang Runping , Lu Yongjun , Cicha Michael , Rahmouni Kamal , Chapleau Mark , Benson Christopher , Abboud Francois
Published: June 2021

The cholecystokinin (CCK) induced satiety signaling of vagal afferents is suppressed in diet‐induced obese mice, which contributes to the increased food intake. In this study we tested the hypothesis that a Ca2+‐activated chloride channel (CaCC) mediates the response of normal intestinal vagal afferent nodose neurons to CCK and that excessively high fat diet (HFD) suppresses the channel activation and leads to obesity. We first used Ca2+ imaging techniques and the whole cell patch‐clamp on cultured nodose ganglia neurons isolated from anesthetized C57/BL6 mice. Application of CCK increased [Ca2+]i in a dose‐dependent manner reaching near maximal level at 10 μM. This dose of CCK also induced a large inward current (36.0±11.0 pA/pF, n=7) that was eliminated with the fast Ca2+ chelator BAPTA (1.0±0.5 pA/pF, n=7). The reversal potential of the current was ~0mV with equal concentration of Cl‐ on both sides of the cell membrane and shifted to a more positive direction after lowering the extracellular [Cl‐]o confirming that it is an outward Cl‐ conductance. Moreover, the CCK‐induced response was reduced significantly from 35.2±3.2 pA/pF in untreated control neurons (n=9) to 13.6±2.0 pA/pF in the presence of niflumic acid (100μM, n=8), a blocker of the CaCC channel (p<0.05). DiI labeled proximal intestinal nodose neurons obtained from control mice responded to CCK with an average current of 24.8±4.9 pA/pF (n=7), whereas the response of labeled neurons from obese mice fed a 60% HFD for 10 weeks was significantly lower at 6.1±2.9 pA/pF (n=7) (p<0.01). Our results demonstrate that a CCK‐sensitive CaCC in proximal intestinal nodose neurons is desensitized in mice fed a high fat diet. This CaCC ionic conductance may be responsible for satiety signaling of vagal afferents and its impairment may contribute to excessive food intake and obesity.

Grant Funding Source: Supported by NIH HL 14388
Multivalent cell specific therapeutics for treatment of metabolic disorders (1108.1)
Authors: Lynch Ron , Weber Craig , Hart Nathaniel , Limesand Sean , Xu LiPing , Morse David , Gillies Robert , Vagner Josef
Published: June 2021

Metabolic Homeostasis is mediated via multi‐organ endocrine signaling. Because many systems are involved, therapeutics often have wide‐ranging side‐effects. We have shown therapeutic specificity can be achieved with agents that simultaneously bind two different receptors that, as a combination, are relatively unique to a given cell type. Bivalent ligands, that target cells within the homeostatic axis, were produced; melanocyte stimulating hormone linked to CCK (MSH/CCK), and Glucagon Like Peptide 1 (GLP‐1) linked to Glibenclamide (Glb, SUR1 antagonist). Binding analyses, assessed with cells expressing the complementary receptor pair, indicate an apparent Kd of ~5 nM for MSH/CCK, ~10nM for GLP‐1/Glb. Constants for the individual domains within the bivalent constructs were 30 nM or greater, and neither ligand bound with high affinity to cells expressing only one of the complementary receptors, indicating that high affinity binding requires both receptors. Once bound, the ligand/ receptor complex is rapidly endocytosed. Both ligands maintain signaling capacity, though the magnitude, sensitivity of 2nd messenger and physiological effects, differ from the constituent monomers. Uniquely, GLP‐1/Glb was found to potentiate insulin secretion at stimulatory glucose concentrations, but inhibit, rather than activate, at non‐stimulatory glucose. Thus, these bivalent ligands exhibit enhanced affinity, cell type specificity and maintain functional efficacy, and thereby hold promise as cell specific therapeutic agents.

Grant Funding Source: Supported by: Juvenile Diabetes Research Foundation, National Institutes of Health, NCI, CA123547
Chronic blockade of the renin‐angiotensin system improves glucose metabolism and pancreatic superoxide dismutase activity in insulin resistant rats (1108.12)
Authors: Rodriguez Ruben , Minas Jacqui , Vazquez‐Medina Jose , Nakano Daisuke , Nishiyama Akira , Ortiz Rudy
Published: June 2021

Blockade of the renin‐angiotensin‐system (RAS) improves pancreatic function, decreases fasting blood glucose (FBG), glucose intolerance, insulin resistance (IR) and delays the onset of type 2 diabetes. However, the timing of the onset of the beneficial effects of RAS blockade is not clear. To assess the timing of the beneficial effects of RAS blockade, we measured fasting blood glucose, glucose intolerance, insulin resistance index (IRI) and pancreatic antioxidant activity after 2 and 11 weeks of angiotensin receptor blocker (ARB) treatment in the following groups of rats: 1) LETO (lean strain‐control), 2) untreated fatty obese‐OLETF, 3) OLETF + ARB (ARB; 10 mg olmesartan/kg/d). OLETF rats exhibited elevated FBG, glucose intolerance and IRI at 9 weeks of age compared to LETO and they worsen with age. Two week treatment with ARB did not decrease FBG, glucose intolerance or IRI, whereas 11 week treatment decreased them by 23%, 26% and 47%, respectively, compared to OLETF. OLETF rats also exhibited decreased pancreatic antioxidant enzyme activity as compared to LETO. Two week treatment with ARB did not increase antioxidant enzyme activity; however, 11 week‐treatment increased 42% superoxide dismutase activity compared to OLETF. These data suggest that the beneficial effects that RAS blockade have on glucose metabolism and pancreatic antioxidant activity are not immediate, but improve with treatment duration.

Grant Funding Source: Supported by NIH NCMHD 9T37MD001480.
Shedding of ACE2 by mature form of ADAM17 is mediated by angiotensin type 1 receptor (1108.14)
Authors: Chodavarapu Harshita , Chhabra Kavaljit , Pedersen Kim , Lazartigues Eric
Published: June 2021

ADAM17 (a disintegrin and metalloprotease) is associated with shedding of ACE2 in diabetic rodent models with overactive Renin‐Angiotensin System. To test the role of ADAM17 in pancreatic ACE2 shedding, C57Bl/6J mice (n=5/group) were fed high fat diet (HFD) for 16 weeks. HFD fed mice showed hyperglycemia (HFD: 150 ±10.7 vs. Control: 103 ±3.6 mg/dl P<0.05) and glucose intolerance (HFD: 43438 ±2803 vs. Control: 35903 ±2075 AU P<0.05). Western blot and Quantitative RT‐PCR showed 2 fold increase in both pancreatic ADAM17 protein and mRNA expression (p<0.05) in HFD mice. This was associated with a 1.5 fold reduction (p<0.05) in pancreatic ACE2 protein and 1.5 fold increase (p<0.05) in mRNA expression in HFD mice. To directly implicate the role of Angiotensin (Ang) II type 1 receptor (AT1R) in ADAM17 expression, an immortalized human pancreatic beta cell line (β‐lox 5) was used. Treatment with 500 nM Ang II resulted in 2 fold increase (p<0.05) in ADAM17 protein expression which was attenuated by losartan (AT1R blocker). Further, transfection of β‐lox 5 cells with hACE2/pcDNA3.1 followed by treatment with an ADAM17 overexpressing adenovirus resulted in 2 fold increase in the pro form (p<0.05) but not the mature form of ADAM17 protein expression and no change in hACE2 expression. In conclusion, our results demonstrate that AT1R‐mediated increase in the mature form of ADAM17 is responsible for the shedding of pancreatic ACE2.

Grant Funding Source: Supported by American Heart Association (110300120A)
Expression of the receptor for proinsulin C‐peptide, GPR146, in the kidney (1108.5)
Authors: Redlinger Lauren , Kolar Grant , Samson Willis , Yosten Gina
Published: June 2021

Proinsulin C‐peptide has been shown to exert protective effects in the microvasculature in both animal models of diabetes and in diabetic humans. In particular, C‐peptide may prevent or reverse diabetic nephropathy. We recently have identified the orphan G protein coupled receptor, GPR146, to be essential for C‐peptide signaling in vitro, using a Deductive Ligand‐Receptor Matching Strategy developed by our laboratory (J Endocrinology 218:B1‐8, 2013). We now report that the human embryonic kidney cell line, HEK293, responded to C‐peptide with an increase in cFos mRNA. To further evaluate the role of GPR146 in C‐peptide signaling, we employed siRNA to selectively knock down GPR146 expression in HEK293 cells and assessed the sensitivity of those cells to C‐peptide. Knockdown of GPR146 completely ablated C‐peptide‐induced cFos expression. When the neuronostatin receptor, GPR107, was selectively knocked down as a control, the response to C‐peptide was unaffected. These data support the hypothesis that GPR146 is involved in C‐peptide signaling in the kidney. The identification of GPR146 as a potential C‐peptide receptor should facilitate future studies examining the physiological relevance of C‐peptide’s action in the kidney.
Expression and function of bile acid receptor TGR5 in glucagon secreting pancreatic α cells: hyperglycemia induced expression of PC1 and release of GLP‐1 in response to activation of TGR5 by bile acids (1108.6)
Authors: Kumar Divya , Mahavadi Sunila , Mirshahi Faridoddin , Grider John , Murthy Karnam , Sanyal Arun
Published: June 2021

We have recently shown that TGR5, a novel G protein coupled receptor, expressed in the pancreatic β cells induces insulin secretion in response to bile acids. Secretion of glucagon from the pancreatic α cells and glucagon‐like peptide 1 (GLP‐1) from the intestinal enteroendocrine cells also regulate insulin secretion. Glucagon and GLP‐1 are processed from the proglucagon by the cell‐specific expression of proconvertase 2 (PC2) and PC1, respectively. AIM. To determine expression and function of TGR5 in pancreatic α cells in normal and hyperglycemia. RESULTS. mRNA and protein expression of TGR5 was demonstrated in α cell line αTC1‐6 and mouse islets. Addition of INT‐777, a selective TGR5 ligand or lithocholic acid (LCA) increased glucagon secretion at low glucose (3 mM) in a concentration dependent manner in αTC1‐6 cells and mouse islets. PC2, but not PC1 was expressed in control αTC1‐6 cells. Treatment of α cells or mice islets with 25 mM glucose for 7 days induced expression of PC1 leading to secretion of GLP‐1 in response to INT‐777 and LCA. Similar release of GLP‐1 was obtained from diabetic db/db mice islets. CONCLUSION. Pancreatic α cells express TGR5 and PC2, and release glucagon in response to bile acids. Hyperglycemia induces expression of PC1 and causes release of GLP‐1. The importance of GLP‐1 in the regulation of insulin secretion underscores the significance of TGR5 as a potential therapeutic target in diabetes.
Fructose stimulates phosphorylation and trafficking of the Na/K/2Cl cotransporter in rat thick ascending limbs (1109.2)
Authors: Ares Gustavo , Kassem Kamal , Ortiz Pablo
Published: June 2021

High fructose consumption affects renal salt handling and is linked to salt‐sensitive hypertension in humans and rodents. The thick ascending limb (TAL) reabsorbs 25% of filtered NaCl via NKCC2. However, the effect of fructose on NKCC2 and TAL NaCl absorption is unknown. We hypothesized that acute and chronic fructose treatment stimulate NKCC2 activity and trafficking in TALs. Acute fructose treatment (20 min) enhanced surface NKCC2 expression (Fructose 1mM= 22 ± 5%, 5mM= 49 ± 10%, 10mM= 101 ± 59%; p<0.05), but did not increase NKCC2 phosphorylation at Ser126, Thre96/101, nor total NKCC2 expression. Fructose (5mM) increased NaCl transport‐related TAL oxygen consumption by 11 ± 3% (p<0.05), and this effect was absent when NKCC2 was blocked by furosemide. In contrast, glucose (5 mM) did not affect surface NKCC2 nor TAL oxygen consumption. When Sprague‐Dawley rats were fed 20% fructose for 1 week, surface‐to‐intracellular NKCC2 increased by 61 ± 23% (p<0.05), Thre96,101 phosphorylation increased by 8 ± 3 fold (p<0.05) but total NKCC2 expression was reduced by 40 ± 9% (p<0.05). We concluded that acute fructose treatment increases surface NKCC2 expression and NaCl transport by TALs. One week on a fructose enriched diet increased NKCC2 trafficking and phosphorylation at Thre96/101. Our data suggest that fructose but not glucose may contribute to salt‐sensitive hypertension by stimulating NKCC2 and NaCl absorption.

Grant Funding Source: 1P01HL090550
Regulation of NCC and the WNK cascade by the circadian clock protein Per1 in murine distal convoluted tubule cells (1109.5)
Authors: Richards Jacob , Ko Benjamin , All Sean , Cheng Kit‐Yan , Hoover Robert , Gumz Michelle
Published: June 2021

Blood pressure and renal function undergo circadian fluctuations. We have shown that the circadian protein Per1 regulates genes involved in sodium (Na) transport in the kidney collecting duct, but has not been investigated in other parts of the nephron. The distal convoluted tubule (DCT) plays a critical role in Na reabsorption in the nephron through the actions of the Na‐Cl co‐transporter (NCC), which is regulated by the with‐no‐lysine kinases (WNKs). Therefore, we wanted to test the hypothesis that Per1 regulates Na transport in the DCT through modulation of NCC, WNK1 and WNK4.

Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of NCC and WNK1, but increased expression of WNK4 in mouse renal cortex. These findings were confirmed by Per1 siRNA and pharmacological blockade of Per1 nuclear entry in mDCT15 cells, a model of the mouse distal convoluted tubule. Measurement of short‐lived hnRNA demonstrated that the effect of Per1 was transcriptional. Chromatin immunoprecipitation experiments demonstrated interaction of Per1 and CLOCK with the promoters of NCC, WNK1, and WNK4; blockade of Per1 nuclear entry abolished these effects. Importantly, NCC activity, as measured by thiazide‐sensitive, Cl‐dependent 22Na uptake, was decreased upon pharmacological inhibition of Per1 nuclear entry. These data demonstrate a role for Per1 in the transcriptional regulation of NCC, WNK1, and WNK4.

Grant Funding Source: Supported by NIH, ASN and AHA
Activation of the AT1 receptor‐beta‐arrestin signaling pathway inhibits NHE3 activity in the renal proximal tubule (1109.8)
Authors: Carneiro de Morais Carla , Oliveira‐Souza Maria , Pessoa Thaissa , Malnic Gerhard , Krieger Jose , Girardi Adriana
Published: June 2021

Previous studies have demonstrated that TRV120023, a novel biased peptide ligand of the angiotensin II type 1 receptor (AT1) which specifically activates the beta‐arrestin mediated signaling pathway, exerts diuretic and natriuretic actions. The present study tested the hypothesis that TRV120023 inhibits NHE3 activity in rat renal proximal tubules (PT) and in a PT cell line (OKP cells). PT NHE3 activity was measured as the rate of bicarbonate reabsorption by in vivo stationary microperfusion whereas Na+‐dependent pHi recovery after an acid load was employed to evaluate NHE3‐mediated H+ extrusion in OKP cells. TRV120023 (10‐7M) significantly inhibited net proximal tubule bicarbonate reabsorption (2.00 ± 0.31 vs. 1.39 ± 0.22 nmol/cm2/s (P <0.001). In OKP cells, Na+‐dependent pH recovery was reduced by TRV120023 in a dose dependent manner. Moreover, experiments performed in the presence of the NHE3 inhibitor S3226 demonstrated that the inhibitory effect on NHE3‐mediated Na+‐dependent intracellular pH recovery decreased approximately 50% after 30 minute‐treatment with TRV120023 (0.18 ± 0.03 vs. 0.09 ± 0.03 pH units/min, P<0.001). The inhibitory effect of TRV120023 on NHE3 activity in OKP cells was blocked by wortmannin, a phosphatidylinositol‐3‐kinase (PI3K) inhibitor but not by the ERK inhibitor U0126. Taken together our results suggest that the diuretic and natriuretic effects of TRV120023 may be, at least in part, due to renal proximal tubule NHE3 inhibition via activation of the AT1R/beta‐arrestin/PI3K signaling pathway.

Grant Funding Source: FAPESP
Vagally‐mediated gastric effects of catecholamines in stressed rats (1110.13)
Authors: Browning Kirsteen , Fries Allison , Travagli R. Alberto
Published: June 2021

Stress exerts vagally‐mediated action to disrupt gastric motility. Preliminary results from our laboratory suggest that brainstem vagal neurocircuits undergo remodeling following stress and previous studies from other groups have shown that the A2 catecholaminergic area is involved in these effects.

The aim of this study was to investigate whether stress modulates the response of vagally‐mediated gastric pathways to catecholamines.

Inhibitory postsynaptic currents (IPSCs) were recorded in neurons of the dorsal motor nucleus of the vagus (DMV) following NTS stimulation and perfusion of the slices with the α2 agonist Uk14304 (0.1‐10μM). In control rats, UK14304 had no effect on 5 of 5 neurons tested (96±3% of baseline amplitude); in contrast, UK14304 decreased the amplitude of IPSC in 12 of 17 rats that underwent homotypic stress for 5 days in a concentration‐dependent manner (91±1%, 78±1% and 53±9% of baseline amplitude in response to 0.1, 1 and 3μM, respectively; P<0.05 vs control). In control rats, brainstem microinjections of the indirect sympathomimetic tyramine (4.5nmoles/60nl) reduced gastric motility to 50±10% of baseline (N=10); conversely, microinjections of tyramine in the brainstem of homotypic stress rats decreased gastric motility to 7±5% of baseline (N=2).

These data suggest that homotypic stress induces an upregulation of catecholaminergic, including α2‐mediated, responses in vagal neurocircuits resulting in an exaggerated vagally‐mediated gastroinhibition.

Grant Funding Source: NIH DK55530, DK78364
Effects of hibernation on gut motility in thirteen‐lined ground squirrels (1110.4)
Authors: Liu Sumei , Brandenburg Cheryl , Eliades Lauren , Erlandson Martin , Cooper Scott
Published: June 2021

Hibernating animals experience dramatic changes in the structure and functions of their digestive system along with other body organ systems. Gut motility helps to propel food along the digestive tract and mix it with digestive enzymes. Despite its importance, changes in gut motility during hibernation have received far less attention than other physiological functions. The aim of this study was to investigate the effects of hibernation on gut motility and neuronal regulation. Thirteen‐lined ground squirrels in summer active, winter torpor, and interbout arousal states were used in the study. Gastrointestinal transit was measured using a charcoal meal method. Immunofluorescence was used to measure the expression of neurotransmitter synthetic enzymes in the enteric nervous system (ENS) of the small intestine. Gastrointestinal transit was significantly slowed down during torpor, but returned to normal level during interbout arousal. Choline acetyltransferase (ChAT) is an enzyme used in the synthesis of acetylcholine, a major excitatory neurotransmitter in the ENS. Nitric oxide synthase (NOS) is an enzyme used in the synthesis of nitric oxide, a major inhibitory neurotransmitter in the ENS. The number of ChAT‐immunoreactive (IR) neurons was significantly decreased during torpor, but NOS‐IR neurons remained unchanged. Thirteen‐lined ground squirrels slow down gastrointestinal motility during torpor. Selective downregulation of ChAT may contribute to gut hypomotility during torpor. Acknowledgement: UW‐L research grant (CB and SL).
Glp2r is required for postprandial glycemic control (1114.3)
Authors: Shi Xuemei , Wen Shixiang , Chacko Shaji , Chan Lawrence , Guan Xinfu
Published: June 2021

Glucagon‐like peptide 2 (GLP‐2) is secreted from enteroendocrine L cells and proposed as a key promoter of intestinal metabolic reprogramming and tissue regeneration, contributing to improving glucose homeostasis and insulin sensitivity after RYGB. Through the gut‐brain‐liver axis, GLP‐2 plays crucial roles in the control of food intake and hepatic glucose production. We showed in Cell Metab (2013) that GLP‐2 receptor (Glp2r) in POMC neurons is required for GLP‐2 to promote glucose homeostasis at the postabsorptive status. However, little is known about the physiological relevance of GLP‐2 in the glycemic control at the postprandial status when GLP‐2 is physiologically secreted. In this study, we first developed an innovative stable isotopic tracer approach to quantify non‐steady postprandial glucose kinetics in mice, which were assessed by the appearance rate of ingested glucose (Ra,ingested) after intragastric feeding U‐13C6‐D‐glucose‐enriched elementary diet; and the appearance rate of endogenous glucose (Ra,endogenous) by iv infusion of 6,6‐2H2‐D‐glucose. We then generated mice with Glp2r deletion in the intestinal epithelium by crossing Glp2r‐floxed mice with villin‐Cre mice. Finally, we show that Glp2r global KO mice display glucose intolerance after an oral GTT and postprandial hyperglycemia after the elementary meal as a result of increased hepatic production of endogenous glucose (Ra,endogenous, attributed to the increased rate of gluconeogenesis) and decreased splanchnic sequestration of ingested glucose (Ra,ingested), pointing to a physiological role of GLP‐2 in postprandial glycemic control. Moreover, villin‐Glp2r KO mice display postprandial glucose intolerance, suggesting that the gut epithelium is a key site for GLP‐2 action to promote postprandial glycemic control. We conclude that Glp2r activation is required for postprandial glycemic control and GLP‐2 secretion augmentation may contribute to improving postprandial hyperglycemia in diabetes.

Grant Funding Source: Supported by the USDA CRIS 6250‐51000‐055
Activation of IKK2‐NF‐κB in adult mouse liver produces a cytoprotective gene expression profile without signs of inflammation, insulin resistance, or fibrosis (1115.4)
Authors: Lu Hong , Lei Xiaohong , Zhang Qinghao
Published: June 2021

The NF‐kB signaling, regulated by IKK1 and IKK2, is activated by various stresses to protect or damage the liver, in context‐specific manners. Two studies of liver‐specific expression of constitutive active IKK2 (Ikk2ca) showed that activation of IKK2‐NF‐kB in developing liver caused inflammation, insulin resistance, and/or fibrosis. The purpose of this study was to understand how activation of IKK2‐NF‐kB in adult liver alters gene expression and pathophysiology. Using Ikk2ca knockin and Alb‐cre mice, we generated mice with adult hepatocyte‐specific activation of IKK2 (Liv‐Ikk2ca). Adult male Liv‐Ikk2ca mouse livers had induction of mRNAs of Ikk2 and classical NF‐kB targets, as well as IKK1, NIK (activator of IKK1), and RelB, but no changes in markers of inflammation or fibrosis (by real‐time PCR). Liv‐Ikk2ca mice had reduction of prooxidative Cyp2e1 and Cyp4a14, induction of antioxidative Nqo1 and Sod2, but no changes of key enzymes in fatty acid oxidation and gluconeogenesis. Regarding nuclear proteins of NF‐kB subunits, Liv‐Ikk2ca mice had moderate increase in p50, strong increase in RelB, but no changes in p65 (by Western blot). In summary, IKK2‐NF‐kB activation in normal adult hepatocytes produces a cytoprotective gene expression profile without signs of inflammation, insulin resistance, or fibrosis, likely due to strong activation of the IKK1‐regulated antiinflammatory alternative NF‐kB pathway.
H1‐antihistamines exacerbate high fat diet‐induced fatty liver disease in wild type but not in apolipoprotein E‐knockout mice (1116.10)
Authors: Vimala Raveendran Vineesh , Kassel Karen , Smith Donald , Cherian Rachel , Reed Gregory , Flynn Colleen , Luyendyk James , Dileepan Kottarappat
Published: June 2021

Activation of histamine receptor 1 (H1R) reduces adiposity in mice, and chronic use of H1‐antihistamines (H1‐AH) increase weight gain in obese patients. Apolipoprotein E (ApoE) plays a major role in lipid transport. We examined the effects of chronic blockade of H1R by cetirizine or fexofenadine, two therapeutic H1‐AH, on fatty liver disease, adiposity and weight gain in wild‐type (WT) and ApoE‐knockout (ApoE‐/‐) mice. Eight week old male WT or ApoE‐/‐ mice were fed a high fat diet, and concurrently treated with equivalent therapeutic doses of either cetirizine or fexofenadine via drinking water for 3 months. H1‐HA treatments increased weight gain, visceral adiposity, liver weight and macrovesicular hepatosteatosis in WT mice, but not in ApoE‐/‐ mice. These changes were independent of food intake. Serum markers of liver injury tended to increase in H1‐HA‐treated WT mice in association with increased macrovesicular hepatosteatosis. Microvesicular hepatosteatosis and lobular inflammation were not affected by H1‐HA treatments in WT or ApoE‐/‐ mice. Increased hepatic steatosis was not associated with changes in the expression of pro‐lipogenic genes SREBP1c or FAS. H1‐HA treatments significantly reduced hepatic ApoE, but not ApoB100, gene expression, and reduced serum triglycerides and VLDL in WT mice. This effect was not seen in ApoE‐/‐ mice. The results suggest that H1‐HA exacerbate HFD‐induced obesity and hepatic steatosis via a mechanism requiring ApoE.

Grant Funding Source: Supported by NIH R01‐HL070101 & 3R01‐HL070101‐04S1, J & E Carey Arthritis Fund & A E Smith Res Fund
Umbelliferone supplementation attenuates alcohol‐induced hepatic steatosis by regulating PPARα‐ and SREBP‐1c pathway in rats (1116.2)
Authors: Sim Mi‐Ok , Lee Hae‐In , Ham Ju Ri , Choi Ra Young , Kim Sung Jin , Seo Kwon‐Il , Kim Myung‐Joo , Lee Mi‐Kyung
Published: June 2021

Umbelliferone (UF) is a 7‐hydroxycoumarin obtained from plants and edible fruits and roots, which has several biological activities including, antitumor, antioxidant and antiangiogenic activity. This study investigated the effects of UF on alcoholic fatty liver and its underlying mechanism. Rats were fed a Lieber‐DeCarli liquid diet with 36% of calories as alcohol with or without UF (0.05 g/L) for 8 weeks. Normal rats were received isocaloric carbohydrate liquid diet. UF significantly improved alcohol‐induced loss of body weight and hepatic lipid accumulation and droplets as well as dyslipidemia. UF significantly suppressed the lipogenic gene expression (SREBP‐1c, CIDEA, FASN and PPARγ) and stimulated the lipolytic gene expression (PPARα, Acsl1, CPT‐1, ACOX and Acca1a) compared to the alcohol control group, which could lead to inhibit fatty acid synthase activity and stimulate CPT and fatty acid β‐oxidation activities in the liver of chronic alcohol‐fed rats. These results indicated that UF attenuated alcoholic steatosis by down‐regulation of SREBP‐1c‐mediated lipogenesis and up‐regulating of PPARα‐medicated lipolysis. Therefore, UF may provide a promising natural therapeutic strategy against alcoholic fatty liver.

Grant Funding Source: Supported by Suncheon Research Center for Natural Medicines
Role of JMJD3 and histone modifications in intermittent hypoxia‐induced neuroinflammation (1120.6)
Authors: Smith Stephanie , Watters Jyoti
Published: June 2021

Microglial phenotypic plasticity enables them to sense and respond to a variety of stimuli. While much of this plasticity involves transcriptional gene regulation that is highly susceptible to epigenetic modulation, little is known about how post‐translational histone modifications regulate microglial activities. We have shown that intermittent hypoxia (IH), a hallmark of sleep apnea, induces microglial inflammation in vivo, but mechanisms underlying IH‐induced microglial activation are unknown. The Jumonji gene family of histone demethylases are hypoxia‐sensitive, and JMJD3 is known to regulate macrophage inflammatory processes, but nothing is known about its regulation of microglial activities, or its role in IH‐induced neuroinflammation. Thus, using in vivo and in vitro models we tested the hypothesis that JMJD3 is necessary for IH‐induced microglial inflammation. In vivo, JMJD3 mRNA is upregulated in microglia immunomagnetically isolated from the cortex and hippocampus of IH‐treated animals, with a time course that correlates with inflammatory gene induction. To test the requirement of JMJD3 in IH‐induced microglial gene expression, we exposed microglial cell cultures to IH in the presence and absence of the small molecule JMJD3 inhibitor GSK‐J4 (7.5μM). IH increased JMJD3 mRNA levels which correlated with a decrease in histone H3 lysine 27 (H3K27) trimethylation, the enzymatic target of JMJD3 activity. IH‐induced IL‐6, IL‐1β, iNOS, and COX‐2 pro‐inflammatory gene expression was blocked in the presence of GSK‐J4, suggesting a critical role of JMJD3 and H3K27 demethylation in IH‐induced inflammation. This is the first study to investigate a Jumonji demethylase in the context of IH, and the first to link IH‐induced microglial inflammation to JMJD3 activities.

Grant Funding Source: Supported by NIH R01 HL111598 and the Graduate Women in Science Ruth Dickie Fellowship
Initial characterization of leptin receptor knockout Dahl salt‐sensitive rats (1121.2)
Authors: McPherson Kasi , White Tiffani , Johnson Ashley , Geurts Aron , Jacob Howard , Garrett Michael , Williams Jan
Published: June 2021

Obesity‐induced metabolic syndrome (MetS) has grown at an alarming rate within the last decade and has been associated with the development of chronic kidney disease (CKD). In the current study, we created a 16 base pair frame‐shift deletion in exon 11 of the leptin receptor gene within the genetic background of the Dahl SS rat (SSLepr‐/‐ strain) using Zinc‐finger nucleases to study the effect of obesity on the progression of renal injury in SS rats. At 6 weeks of age, body weight was 45% higher in the SSLepr‐/‐ strain compared to SSWT rats and remained markedly elevated throughout the entire study (22 weeks of age). The SSLepr‐/‐ strain exhibited impaired glucose tolerance and increased plasma insulin levels as early as 6 weeks of age suggesting insulin resistance while SSWT rats did not. However, glucose levels remained in the normal range throughout the course of the study. At 22 weeks of age, the SSLepr‐/‐ strain developed severe systolic hypertension compared to the values observed in SSWT rats (242±17 vs. 158±6 mmHg, respectively). After 22 weeks of age, proteinuria increased to 618±36 mg/day in the SSLepr‐/‐ strain while it only increased to 136±28 mg/day in SSWT rats. The SSLepr‐/‐ strain exhibited more glomerular injury and renal fibrosis compared to SSWT rats. Overall, these data indicate that the SSLepr‐/‐ strain may be a useful model to study signaling pathways that play a role in CKD associated with MetS.

Grant Funding Source: Supported by P20GM104357
Research community driven development to genetically modify rat models for heart, lung, blood and sleep disorders (1121.3)
Authors: Lazar Jozef , Dwinell Melinda , Geurts Aron , Mattson David , Jacob Howard
Published: June 2021

Rats have a long history of yielding critical knowledge about the cardiovascular and pulmonary systems, including mechanisms of blood pressure regulation in health and disease and remain a major model system for discovery, validation, defining physiological mechanisms and testing of new compounds to improve human health. As noted in the National Heart, Lung and Blood Institute (NHLBI) Strategic Plan, there is a need for animal models to validate human variants. To address this, we will generate 200 gene modified rat strains in the next 5 years using the most economical gene editing technologies (TALEN, CRISPR/Cas 9 and a few others in development). This resource minimizes the prohibitive cost for individual laboratories to develop a single strain. Strain development is prioritized by our External Advisory Board, in consultation with NHLBI to maximum the utilization of the resources to benefit the NHLBI scientific community. Furthermore, the program will provide not only gene modified animals, but also cryopreservation, and in some cases phenotyping upon a request of investigator. Two competitive application deadlines per year are implemented and can be submitted to: mcwcustomrats@mcw.edu. Additional information is available at: http://rgd.mcw.edu/wg/gerrc. For genes not within the NHLBI portfolio or priority, we can offer economical model development with straight forward licensing and maximum degrees of freedom to work.

Grant Funding Source: Supported by NIH NHLBI R24HL114474
Bestrophin‐3 is expressed in the mouse intestine (1122.1)
Authors: Eliasson Emilie , Golubinskaya Veronika , Nilsson Holger
Published: June 2021

Bestrophins, named after their association with Best’s disease (vitelliform macular dystrophy) are transmembrane proteins that function as calcium‐activated chloride channels (CaCC's), but may also play a role in cell survival. Bestrophins exist in several isoforms. Bestrophin‐3 is a cyclic‐GMP‐regulated CaCC in vascular smooth muscle. Its role in the intestine has not been studied, but another isoform, bestrophin‐2, is present as a chloride or bicarbonate transporter in the goblet cells of the colon. We have here determined the expression of bestrophin‐3 in the intestine of C57BL/6 mice using immunohistochemistry and Western blotting.

Bestrophin‐3 was detected in varying proportions in goblet cells all along the intestine, and also in glia‐like cells in the entire myenteric plexus, as well as in stromal cells mainly in the colon submucosa. Staining of cells in the myenteric plexus was also detected in P10 pups.

Thus bestrophin‐3 is expressed in several specific locations along the intestine. Its function in these locales remains to be determined.
Acute tetrahydrobiopterin restores endothelial function in patients with COPD (1123.1)
Authors: Harris Ryan , Seigler Nichole , Halasan Karen , Livieratos Speros , Dillard Thomas , Bass Leon
Published: June 2021

Chronic obstructive pulmonary disease (COPD), although a disease of the lungs, is accompanied by systemic oxidative stress and inflammation, both of which contribute to reductions in nitric oxide (NO) bioavailability. Tetrahydrobiopterin (BH4) is an essential cofactor for eNOS and subsequent NO bioavailability. This study sought to determine if acute ingestion of BH4 improves endothelial function in patients with COPD. 18 patients with COPD and 15 healthy controls participated in this study. The flow‐mediated dilation (FMD) test was performed at baseline in all subjects, and 3 hours following ingestion of either BH4 (5 mg/kg of Kuvan®) dissolved in 4oz of apple juice or placebo only in patients. No differences in sex or race were observed between groups. Age (56±2 vs. 57±2 yrs.), BMI (31.5±1.9 vs. 26.6±1.6 kg/m2), and MAP (93.9±2.8 vs. 88.0±2.7 mm Hg) were all similar (p>0.05) between patients and controls, respectively. FEV1 was significantly lower in patients (56±4 vs. 103±4 %predicted) compared to controls. At baseline, FMD was lower (p=0.046) in patients (4.9±0.5%) compared to controls (6.9±0.9%). Acute BH4 treatment significantly improved FMD (α3.4±0.6%; p<0.001) in patients; however, no change (α‐0.2±0.6%; p=0.703) in FMD was observed following placebo. Findings from this study demonstrate that a single dose of BH4 transiently restores endothelial function in patients with COPD to the values observed in controls. Supported by the American Heart Association (RAH).

Grant Funding Source: Supported by AHA 10SDG305006 (RAH)
Inhibition of endoplasmic reticulum function in PVN by thapsigargin increases neuronal excitability and sympathetic nerve activity (1125.4)
Authors: Larson Robert , Gui Le , Chapp Andrew , Huber Michael , Zhu Jianhua , Cheng Zixi , Shan Zhiying , Chen Qing‐Hui
Published: June 2021

Control of sympathetic outflow by paraventricular nucleus (PVN) is partially mediated through a monosynaptic axon projecting to the rostral ventrolateral medulla (PVN‐RVLM). Here we investigated the role of thapsigargin (TG), the endoplasmic reticulum (ER) Ca2+ ATPase inhibitor, in regulating the excitability of PVN‐RVLM neurons under brain slice preparation. In current‐clamp recordings, graded current injections evoked graded increases in spike frequency. Maximum discharge was evoked by +200 pA injections and averaged 22 ± 2 Hz (n=7) and was significantly greater (P < 0.05) in the presence of TG (0.5 µM) (31 ± 3 Hz, n=9). The effect of ER Ca2+ store depletion in regulating renal SNA (RSNA) and mean arterial pressure (MAP) was assessed by bilateral PVN microinjection of TG in anesthetized rats. PVN injection of TG (0.15, 0.3 0.75 and 1.5 nmol/100nl) increased RSNA and MAP in a dose‐dependent manner. Maximum increases in RSNA and MAP elicited by PVN TG (0.75 nmol/100nl; n=3) were 105 ± 31% and 18 ± 11 mmHg respectively. Pre‐treatment with CyPPA (5 nmol/100 nl), a small conductance, Ca2+ activated K (SK) channel activator (n=3), attenuated the PVN TG evoked sympathoexcitatory and pressor responses (7 ± 7% for RSNA and 4 ± 7 mmHg for MAP). PVN CyPPA did not significantly alter baseline RSNA and MAP. Our data indicate that inhibition of ER function with TG enhances in vitro excitability of PVN‐RVLM neurons and increases in vivo RSNA and MAP. The effect of TG on the excitability of PVN neurons and SNA through ER Ca2+ store depletion may involve reduced SK channel activity.

Grant Funding Source: Supported by AHA10SDG2640130
Orexin A in the ventrolateral preoptic area increases feeding and spontaneous physical activity (1126.1)
Authors: Rezaimalek Almira , Teske Jennifer
Published: June 2021

The brain regulates behaviors influencing energy balance such as arousal, feeding and spontaneous physical activity (SPA), which is all activity excluding formal exercise yet mechanisms are unclear. Orexin A (OXA), an endogenous neuropeptide produced in the lateral hypothalamus (LH), increases arousal, SPA and feeding. Orexin innervates the ventrolateral preoptic area (VLPO). Activation of the VLPO inhibits wakefulness and promotes sleep but whether OXA in VLPO modulates SPA and feeding is unclear. We hypothesized that OXA in VLPO would increase feeding and SPA. To test this, male Sprague‐Dawley rats were implanted with a cannula targeting VLPO. OXA (15, 31, 62, 125 pmol/0.5uL) or artificial cerebrospinal fluid (acsf) was injected in VLPO. SPA was measured for 4.5 hours post‐injection by infrared beam break. In a separate group of rats with VLPO cannula, the feeding response to 125 pmol/0.5uL or acsf was measured 2h post‐injection. OXA dose‐dependently increased distance traveled and speed but not vertical movement for 3h post‐injection. Distance traveled was positively correlated to speed and OXA significantly increased food intake. These data suggest that OXA from the LH acts in the VLPO to modulate behaviors such as feeding and SPA in addition to arousal. Identifying the neural mechanisms underlying feeding and SPA would have profound implications for treating obesity and insomnia.

Grant Funding Source: Supported by Department of Veterans Affairs
Regulation of leptin receptor expressing neurons in the brainstem by TRPV1 (1126.11)
Authors: Zsombok Andrea , Jiang Yanyan , Anwar Imran , Rezai‐Zadeh Kavon , Muenzberg‐Gruening Heike
Published: June 2021

The central nervous system plays a critical role in the regulation of feeding and whole body metabolism via controlling the autonomic output to the visceral organs. Activity of the parasympathetic neurons in the dorsal motor nucleus of the vagus (DMV) determines the vagal tone and thereby modulates the function of the organs. Due to the increased prevalence of eating and metabolic disorders, there is a constant search for new compounds, receptors or modulators to alter the activity of autonomic output and thus influence feeding or metabolism. TRPV1 (transient receptor potential vanilloid type 1) plays a pivotal role in synaptic regulation and here, we tested the hypothesis that TRPV1 regulates the activity of leptin receptor (LepRb) expressing DMV neurons. First, whole‐cell patch‐clamp recordings were performed to determine the effect of TRPV1 activation on LepRb neurons. Capsaicin, a TRPV1 agonist increased the frequency of mEPSCs in 50% of the LepRb neurons. Next, we identified stomach‐projecting LepRb neurons in the DMV using the transsynaptic retrograde viral tracer, PRV‐614. Activation of TRPV1 increased excitatory neurotransmission to stomach‐related LepRb neurons. These data demonstrate that TRPV1 is involved in the regulation of a subpopulation of LepRb DMV neurons via presynaptic mechanism and suggest a potential interaction between TRPV1 and leptin signaling in the DMV.

Grant Funding Source: NIH P30GM103337, Tulane Pilot Project