bluuebunny/crossref_metadata_2025 huggingface dataset:
Duration‐dependent effects of IH‐stimulated Hif1a signaling on adult hippocampal neurogenesis
Authors: Nallamothu Thara , Khuu Maggie A. , Garcia Alfredo J.
Published: June 2021

Intermittent hypoxia (IH), is a hallmark consequence of sleep apnea, a common untreated disorder associated with impaired neurological health. We have recently demonstrated that thirty days of IH impairs long term potentiation and reduced the proportion of neurons generated during adult neurogenesis. Hypoxia Inducible Factor 1a (HIF1a) is a transcription factor important to stem cell development that is regulated by the state of oxygenation. The objective of this on‐going study is to characterize how the duration of IH exposure influences the generation of adult‐born neurons and to determine the role of IH‐dependent HIF1a signaling in the generation of adult‐born neurons in the hippocampus. We hypothesize that IH has duration dependent influence over neurons generated through adult neurogenesis and involves HIF1a dependent signaling. We used a cre‐loxP transgenic strategy to birth label a discrete cohort of stem cells that experience room air for 30 days (control), 10 days of IH +20 days of recovery in room air (IH10+20RA), or 30 days of IH (IH30) in wild‐type reporter mice (Ai27) and in Ai27‐HIF1a+/flox mice. In wild‐type mice, exposure to IH10+20RA led to higher proportion of birth‐labelled neurons compared to the control (+19% ± 5%, n=6) while the proportion of neurons following IH30 decreased (−40% ± 10%, n=4). In Ai27‐HIF1a+/− mice, the proportion of birth‐labeled neurons was similar between control and IH10+20RA groups. However, following IH30 the proportion of birth‐labeled neurons was markedly decreased when compared to control in Ai27‐Hif1a+/− and to the IH30 wild‐type group. Dendritic complexity, as determined by Sholl analysis, was similar among labeled neurons in both wild‐type and Ai27‐HIF1a+/− groups suggesting that neither IH nor IH‐dependent HIF1a signaling influences dendritic complexity. These findings indicate that exposure duration is factor impacting how IH affects the generation of adult‐born neurons through a HIF1a signaling shaping how IH influences, associated with sleep apnea affects the generation of adult‐born neurons.

Support or Funding Information

This work was supported by NIH R01 NS10742101 (AJG).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Environmental Enrichment Dampens the Increase in Glucocorticoid Receptors in the Hippocampus Caused by Endometriosis
Authors: Rivera‐Lopez Leslie , Appleyard Caroline B. , Torres‐Reveron Annelyn
Published: June 2021

Endometriosis is a disorder in which the development of uterine‐lining tissue is found outside the uterine cavity causing infertility, pain and stress. Previous studies have shown that stress can negatively affect the progression of the disease. Under the control of the hypothalamic pituitary adrenal (HPA) axis, circulating glucocorticoids regulate immune and stress responses by binding to the glucocorticoid receptors (GR). Our objective was to determine if GR is altered in the brain hippocampal area of rats with endometriosis. We focused on the hippocampus as this is one of the main brain areas that display GR. In addition, hippocampal GR play a significant role in HPA axis hyperactivity. Endometriosis was induced in female rats (n=16) by suturing uterine horn tissue next to the intestinal mesentery and the disease was allowed to progress for 60 days, during which rats were exposed to either environmental enriched conditions (increased social groups, larger enclosures, toys and nesting) or normal housing conditions. Sham rats (n=8) had only sutures in the intestinal mesentery and were exposed to normal housing conditions. At the time of sacrifice, endometriotic vesicles, uterus and brains were collected. A single labeling with DAB immunohistochemistry was performed to analyze the levels of GR in the Cornus amon (CA1 and CA3) and Dental Gyrus (DG) of the hippocampus. We have previously documented that environmental enrichment can significantly reduce the progression of endometriosis resulting also in decreased GR expression within endometriotic vesicles compared to nonenriched rats. Results show that rats with endometriosis had 60% increased GR integrated density in CA1 and DG regions of the rat hippocampus compared to sham controls. Non‐enriched animals had double the integrated density of GR in the dorsal CA3 compared to sham controls and this increase was dampened by environmental enrichment. Our data suggest that interventions that decrease stress can provide some benefit to return GR activity to baseline levels in both, peripheral tissues and also in brain structures involved in stress regulation.

Support or Funding Information

NIH‐K07AT008027

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Immune System Changes in a Rat Model of Maternal Vertical Sleeve Gastrectomy
Authors: Spann Redin A , Taylor Erin B , Himel Alexandra R , Grayson Bernadette E
Published: June 2021

Women undergoing metabolic surgery for weight loss see many improvements in health, including reduced adiposity and treatment of type 2 diabetes. Fertility is also improved by bariatric surgery and the number of large for gestational day births are reduced. Unfortunately, fetal demise and low birth weights are more prevalent following bariatric surgery, and children born small for gestational age are at an increased risk of obesity as adults. We have replicated these findings in a rat model of vertical sleeve gastrectomy (VSG), however, the origin of these poor outcomes in offspring remains unknown. We have previously reported a reduction in plasma T lymphocytes in pregnant female VSG rats compared to pregnant Sham surgery rats, and increased circulating Immunoglobulin G (IgG) protein. We also reported in the placenta increased mRNA levels of interleukin 1 β (IL1B), interleukin 1 receptor antagonist, and matrix metalloproteinase 9 in VSG animals compared to Shams as well as increased TUNEL positive cells indicating apoptosis. We have currently expanded our work in the placenta, finding that mRNA for B cell marker, PTPRC, and cytotoxic T cell marker, CD8A, is elevated in VSG dams. In postnatal day 21 (PND21) pups, we measure lower circulating B lymphocytes in both males and females born to VSG dams compared to pups born to Sham dams, as well as elevated total IgG, while T cells and monocytes were not altered. In the pup hypothalamus at PND21, IL1B mRNA is elevated and this persists until PND120 in males fed chow diet. Transcription of other pro‐inflammatory cytokines such as interleukin 6 and tumor necrosis factor alpha, is not altered in the hypothalamus. These data suggest that VSG surgery alters the immune system in utero and these changes may affect immune health postnatally. Future work will determine what signalling pathways transduce this immune response and if they contribute to low birth weight and metabolic syndrome in offspring.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Contribution of basolateral amygdala‐dorsal striatum pathway in stress
Authors: Kondev Veronika , Patel Sachin
Published: June 2021

While the dorsal striatum (DS) has been historically heavily implicated in habit formation and goal‐directed learning, the role of the DS in stress‐induced effects remains unknown. It has been shown that stress biases rodents towards using a DS‐dependent form of memory. We hypothesize that an understudied input from the basolateral amygdala (BLA) to the DS may play a role in stress‐induced changes in habit and fear learning. Using optogenetics, we analyzed BLA inputs to the DS with whole‐cell patch clamp, focusing specifically on the endocannabinoid system due to the abundant expression of the endocannabinoid receptor (CB1) in the DS. We showed that the pre‐pulse ratio was significantly increased in stressed mice, suggesting decreased activity twenty‐four hours after stress. In vivo fibre photometry data was also collected to further explore the role of the DS before and after stress. Furthermore, endocannabinoid agonists and antagonists were administered to behaviorally and molecularly assess the contribution of endocannabinoid signaling in the DS.

Support or Funding Information

T32 Training Grant ‐ Veronika KondevMH107435

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of 1% Mouse Serum on the Catalase‐ and Superoxide Dismutase‐Mimetic Activity of Custom‐Synthesized Cerium Oxide Nanoparticles
Authors: Estevez Ana Y. , Stadler Brad , Erlichman Joseph S.
Published: June 2021

Due to their potent antioxidant activity, cerium oxide nanoparticles (CeNPs) are being investigated as a potential therapeutic for neurodegenerative diseases. CeNPs are small, regenerative and display both catalase‐ and superoxide dismutase‐mimetic activity. However, toxic effects of CeNPs in biological systems have also been reported. Our working hypothesis is that the synthetic and/or biological identity of CeNPs could account for these disparate reports. In particular, the biological identity of CeNPs can be influenced by serum proteins that adsorb onto the nanoparticle surface. This can then impact the catalytic activity as well as pharmacokinetics properties of CeNPs, potentially contributing to toxic effects in an organism. The aim of the present study was to investigate the effects of serum on the catalytic activity of CeNPs. Custom‐synthesized CeNPs were incubated with or without 1% mouse serum for 30 minutes. CeNPs were then spun in an ultracentrifuge at 434, 513 x g for one hour in order to separate the supernatant (SUP) and pellet (PEL) fractions. We used commercially available enzymatic assay kits to assess the catalase‐ and superoxide dismutase (SOD)‐mimetic activity of each fraction. We also assayed oxidase activity, which could be indicative of promiscuous toxic activity. We observed that pre‐incubation with 1% serum did not significantly impact the catalase‐ or SOD‐mimetic activity of the CeNPs. On the other hand, serum pre‐incubation resulted in significantly enhanced oxidase activity that, upon further analysis, was revealed to be an artifact of the serum rather than attributable to promiscuous CeNP activity. In summary, we demonstrate that pre‐incubation with serum does not negatively affect the antioxidant activity of CeNPs, an important step in continuing to develop CeNPs as neuroprotective agents in oxidative stress diseases.

Support or Funding Information

Supported by St. Lawrence University and Cerion, LLC.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Systemic Administration of Serotonin Reduces the Excitability of the Raphé Pallidus‐Brown Adipose Tissue Sympathetic Nerve Pathway
Authors: Mota Clarissa M. D. , Madden Christopher J. , Morrison Shaun F.
Published: June 2021

Activation of 5‐HT1A receptors in the raphé pallidus (RPa) inhibits the skin cooling‐evoked increases in brown adipose tissue (BAT) sympathetic nerve activity (SNA). Our aim was to determine whether intravenous (iv) 5‐HT would affect BAT SNA and if 5‐HT1A receptors in the RPa play a role in mediating any of the effects of iv 5‐HT. In urethane/α‐chloralose anesthetized, neuromuscularly blocked, ventilated rats, electrical stimulation (100–150 μA, single 1 ms pulses) of the RPa evoked compound potentials in BAT SNA. Iv infusion of 5‐HT (500 μg/h) reduced the amplitude of the RPa‐evoked compound potentials in BAT SNA. Skin cooling‐evoked increases in BAT SNA were also inhibited by the iv infusion of 5‐HT, and by an iv bolus of the 5‐HT1A/7 receptor agonist, 8‐OHDPAT (100 μg in 50 μl). Nanoinjection of the 5‐HT1A receptor antagonist, WAY100635 (10 mM, 100 nl), in the RPa completely prevented the iv 8‐OHDPAT‐induced inhibition of BAT SNA, but not that following iv 5‐HT. In conclusion, iv 5‐HT reduces the excitability of the sympathetic pathway from the RPa to BAT, but this effect is not mediated by activation of 5‐HT1A receptors in the RPa. In contrast, activation of 5‐HT1A receptors in the RPa plays a key role in the inhibition of cold‐evoked BAT SNA by iv administration of 8‐OHDPAT.

Support or Funding Information

Supported by São Paulo Research Foundation (FAPESP) grant #2017/24232‐9 (C.M.D.M.) and by NIH R01‐NS091066 (S.F.M.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Leptin‐induced neuroinflammatory effects in the hypothalamic neurons: role of Sirt1 and FoxO1
Authors: Zheng Hong , Liu Xuefei
Published: June 2021

Accumulated evidence indicates that central nervous system signaling pathways may contribute to the etiology and pathogenesis of obesity‐induced hypertension. Hypothalamic leptin‐mediated signaling may contribute to the exaggerated sympathoexcitation in obesity. Leptin can influence food intake, energy expenditure and sympathetic nerve activity via hypothalamic pro‐opiomelanocortin (POMC) neurons in the arcuate nucleus (ARCN) of hypothalamus. The aim of the study was to determine the effects of leptin on the neuroinflammation‐mediated high sympathetic nerve activity in obesity. We used hypothalamic neuronal cell mHypoA‐POMC (CLU500) and microglial cell (SIM‐A9) to observe pro‐inflammatory and anti‐inflammatory cytokines levels after 24 hours leptin treatment. We found that leptin incubation increased the levels of pro‐inflammatory cytokines interleukin 6 (IL‐6), interleukin 1β (IL‐1β), and tumor necrosis factor alpha (TNFα) in both POMC and microglial cells. Leptin treatment also reduced the level of lipoxin A4, a potent anti‐inflammatory bioactive lipid formed from arachidonic acid in both cells. Knockdown nutrient‐sensing enzyme sirtuin 1 (Sirt1) by siRNASirt1 significantly attenuated the levels of leptin‐induced pro‐inflammatory cytokines in the POMC and microglial cells. On the contrary, knockdown transcription factor forkhead box protein O1 (FoxO1) by siRNAFoxO1 significantly enhanced leptin‐induced pro‐inflammatory cytokines. Further, in vivo study, we found significant increased protein expressions of IL‐6, IL‐1β and TNFα in the ARCN in 12 weeks high fat induced obese rats. siRNASirt1 reduced leptin‐mediated renal sympathetic nerve activity in obese rats. The study suggests that enhanced leptin‐mediated sympathetic activation in obesity may via hypothalamic neuroinflammatory pathway. We propose Sirt1 and FoxO1 in hypothalamus are key regulators of leptin signaling pathway contributed to the over sympathetic activation in obesity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long‐term consequences of acute kidney injury during pregnancy
Authors: Szczepanski Jamie , Spencer Shauna‐Kay , Griffin Ashley , Bowles Teylor , Wallace Kedra
Published: June 2021

OBJECTIVE

Women with severe preeclampsia and HELLP syndrome (sPE/HELLP) are more likely to develop acute kidney injury (AKI) compared to women without sPE/HELLP. AKI during pregnancy is associated with rates of maternal mortality and fetal loss that range from 30–60%. Women with a history of sPE/HELLP are more likely to develop hypertension, and have higher incidences of chronic kidney disease, end stage renal failure and cognitive failure compared to women with histories of normal pregnancies. We tested the hypothesis sPE/HELLP leads to brain damage during pregnancy which is exacerbated by AKI.

STUDY DESIGN

On gestational day (GD) 12, mini‐osmotic pumps infusing sFlt‐1 and sEng were placed into rats to induce HELLP. A subset of HELLP and NP rats underwent bilateral renal ischemia‐reperfusion surgery for 45 minutes on GD18 to create AKI. After rats had recovered, all rats were placed in metabolic cages for 24hrs. Mean arterial pressure was measured on GD19 followed by Evan's blue infusion to assess blood brain barrier permeability, or tissue collection and euthanization.

RESULTS

During pregnancy HELLP+AKI rats had a significant decrease in urine output compared to NP rats (1.4±0.28 vs. 0.48±0.12mL/hr; p=0.01). MAP was significantly increased in HELLP rats (p<0.0001) and HELLP+AKI rats (p=0.03) compared to NP rats. Kidney weight was also significantly increased in NP+AKI compared to NP rats (p=0.005) and in HELLP+AKI compared to HELLP rats (p=0.003). Urine creatinine levels were significantly lower in all groups of rats compared to NP rats (p=0.01). NP pups were significantly larger compared to HELLP (p=0.006), NP+AKI (p=0.0002) and HELLP+AKI (p=0.0005) pups. Rats with HELLP had increased blood brain barrier permeability in the brainstem/cerebellum compared to NP rats (p=0.04). Similar results were seen in NP+AKI (p=0.04) and HELLP+AKI (p=0.01) rats compared to NP rats. Whole brain water weight was significantly increased in HELLP+AKI rats compared to NP rats (p=0.03).

CONCLUSION

The results from the current study suggest that AKI during pregnancy contributes to oliguria, hypertension, decreased urine creatinine, maternal brain injury and reduced pup size. In the setting of HELLP+AKI brain injury was worsened compared to rats with just AKI or HELLP syndrome. Studies are currently ongoing to examine the progression to chronic kidney disease and cognitive failure in rats with a history of sPE/HELLP with and without AKI during pregnancy.

Support or Funding Information

Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number P20GM121334. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TRPC6 Inactivation Does Not Protect Against Diabetic Kidney Disease in Streptozotocin (STZ)‐treated Sprague‐Dawley Rats
Authors: Khayyat Naghmeh Hassanzadeh , Kim Eun Young , Dryer Stuart E
Published: June 2021

Glomerular TRPC6 channels are up‐regulated in rodent models of diabetes mellitus, but the role of TRPC6 in driving progression of experimental diabetic nephropathy is controversial. We recently used CRISPR/Cas9 methods to generate Sprague‐Dawley rats with a deletion in exon 2 of the Trpc6 gene that renders the channels non‐functional [1]. In addition, we observed that animals lacking functional TRPC6 were protected in the chronic puromycin aminonucleoside (PAN) model of acquired FSGS [1]. In the present study, 300‐gram adult Trpc6del/del rats and their wild‐type Trpc6wt/wt littermates were examined in the STZ model of type‐1 diabetes using protocols approved by the University of Houston IACUC committee. In our experiments, diabetes was induced by a single intraperitoneal injection of STZ, while controls received vehicle. Animals were not treated with insulin. STZ‐treated rats with severe hyperglycemia (>450 mg/dl) by 5 days after treatment were used for analysis and were monitored for the next 10 weeks. At 4 and 10 weeks after STZ treatment, compared to vehicle‐treated controls, STZ‐treated rats had severe hyperglycemia (745 ± 144 mg/dl, mean ± SD at 10 weeks). They also had elevated plasma triglycerides and total cholesterol, and failed to gain weight. STZ‐treated rats also exhibited markedly increased urine albumin excretion, increased blood urea nitrogen (BUN), and increased plasma creatinine and decreased creatinine clearance, indicating marked declines in renal function in diabetic rats. Two‐way ANOVA analyses of these datasets revealed no differences between Trpc6wt/wt and Trpc6del/del rats in any of these parameters. There was no time in the course of the experiment where we obtained functional data suggesting a protective effect of TRPC6 inactivation on blood glucose, or kidney function in STZ diabetes. Histological analysis carried out 10 weeks after STZ treatment revealed marked glomerular and tubulointerstitial disease in STZ‐treated rats. Mesangial expansion was the prominent feature in glomeruli of STZ‐treated animals, and glomeruli were less disrupted than we previously observed in the chronic PAN model [1]. Blind semi‐quantitative analysis of glomerular histology revealed a significant effect of STZ but no protective effect of TRPC6 inactivation on glomerular pathology. In summary, TRPC6 inactivation does not prevent declines in renal function and appears to have no protective effect in the STZ model of type‐1 diabetes in Sprague‐Dawley rats. This is in marked contrast to our previous work in chronic PAN nephrosis, where TRPC6 inactivation preserves renal function and histology.

Support or Funding Information

Supported by NIH grant R01‐DK104708.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Urinary prorenin/renin and soluble prorenin receptor excretion in Type 1 and Type 2 diabetes
Authors: Ramkumar Nirupama , Stuart Deborah , Kohan Donald
Published: June 2021

Background

Activation of the intra‐renal, rather than systemic, renin angiotensin system (RAS) has been suggested to play a role in the development of diabetic kidney disease. In this study, we examined plasma and urinary prorenin/renin levels and urinary soluble prorenin receptor (sPRR) levels in mice with early Type 1 and Type 2 diabetes.

Methods

Male Akita (Type 1 model bearing a mutated Ins2 gene) and db/db (Type 2 model bearing a mutated LepR gene) mice were studied from 10–18 weeks age and metabolic balance studies conducted at 4 week intervals on a normal salt diet.

Results

Diabetic mice were hyperglycemic (Akita: 479 ± 31, db/db: 314 ± 60, control: 152 ± 12 mg/dL), polyuric (Akita: 22.2 ± 3.9, db/db: 10.9 ± 2.3, controls 1.6 ± 0.1 ml/day) and polydipsic (Akita: 24.6 ± 3.4, db/db: 11.5 ± 2.4, controls: 4.2 ± 0.5 ml/day) at 10 weeks which continued until 18 weeks. Compared to controls, Akita mice had lower body weight (23.4 ± 0.6 vs controls: 29.2 ± 0.3 grams) while db/db mice had higher body weight (51.9 ± 0.3 g/day) at 10 weeks which continued until 18 weeks. Compared to controls, urinary angiotensinogen and total prorenin/renin levels were increased in Akita mice at 10 weeks and remained elevated at 14 and 18 weeks of age (Table 1); these increases were proportionally greater than changes in urinary albumin excretion. Urinary sPRR excretion was markedly increased in Akita mice at 14 and 18 weeks. In contrast, db/db mice had higher urinary albumin excretion, urinary angiotensinogen and urinary prorenin/renin excretion relative to controls at all 3 time points but undetectable urinary sPRR excretion until 18 weeks. Plasma renin concentration was similar between the controls and diabetic mice at all time points. There was mild glomerular hypertrophy at 18 weeks of age in db/db but not Akita mice.

Conclusion

There is early activation of the intra‐renal but not systemic RAS in Type 1 and 2 diabetes. However, the pattern of intra‐renal RAS component activation appears to differ between Type 1 and 2 diabetes.

Support or Funding Information

University of Utah Internal Medicine Academic Excellence Seed Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sensory Receptors Are Found in a Non‐sensory Organ: The Bladder
Authors: Smith Tilmira A. , Berkowitz Dan E. , Pluznick Jennifer L.
Published: June 2021

Recent research has found that “sensory” receptors (including olfactory receptors (ORs), taste receptors (TRs), and opsins) play functional roles throughout the body. However, the expression of these receptors has not yet been carefully examined in the bladder. In this study, we utilized customized Taqman low density gene array cards to perform quantitative PCR and identify novel sensory receptors in mouse bladder tissue. We extracted RNA from murine bladder (2 male, 2 female), and then screened for the presence of sensory receptors using custom Taqman arrays which included probesets for 43 ORs, 35 bitter TRs, 3 TRs for umami/sweet, 5 opsins, and 7 G‐proteins and/or accessory proteins associated with sensory signaling pathways, as well as a Gapdh control. Using these arrays, we found the expression of 30 sensory transcripts in all male and female bladder samples, with 11 receptors being relatively well‐expressed (Ct values<~30). The 11 highly expressed receptors included 2 opsins (Opn1sw, Opn3), 3 ORs (Olfr558, Olfr99, Olfr78), 5 TRs (Tas1r3, Tas2r143, Tas2r135, Tas2r108, Tas2r126), and the G protein for olfaction (Gnal). We did not note any sex differences, and the top hit (Opn1sw) was the same for all four arrays. Notably, two of the ORs found in the bladder (Olfr78, Olfr558) are known to respond to bacterial metabolites, which may be relevant in the setting of cystitis/urinary tract infections (UTIs). In the future, determining the role of these novel bladder sensory receptors has the potential to enhance our understanding of bladder function, and may lead to novel treatments for conditions such as cystitis/UTIs.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of Angiotensin II on Chronic Renal Disease: Participation of NADPH Oxidase Enzymes, Endoplasmic Reticulum Stress and Tubular Na+ Transporters
Authors: Lins Bruna Bezerra , Casare Fernando Augusto Malavazzi , de Souza Maria Oliveira
Published: June 2021

Introduction

Chronic kidney disease (CKD) associated with hypertension is a serious disease of high incidence in the health system, and its progression may be associated with renal disorders induced by angiotensin II (Ang II). Thus, the objective of this study was to investigate the mechanisms by which Ang II participates in renal injury associated with CKD, considering the contribution of the NADPH oxidase enzymatic complex to the generation of reactive oxygen species (ROS) and the associated factors with of endoplasmic reticulum (ER) stress, as well as the changes in the tubular sodium (Na+) transporters expression.

Methods

Male Wistar rats weighting between 150 and 200 g were treated with Ang II (200 ng/kg/min, by osmotic mini pump, model 2006, ALZET) for 42 days and/or losartan (10 mg/kg/day per gavage) for 14 days and organized into four groups: Control; Treated with Ang II; Treated with losartan and co‐treated with Ang II / losartan. During treatment, the tail blood pressure was evaluated. After treatment, the total renal tissue was used for gene expression analyzes by real‐time polymerase chain reaction (qPCR) and protein expression by western blotting. Immunohistochemistry and immunofluorescence staining were evaluated in the kidney cortex. Statistical analysis was assessed by one‐way ANOVA followed by the Bonferroni's test. Significance was assumed at p<0.05.

Results

The results indicate that in Ang II chronic treatment induced significant increase in mRNA expression for Nox2 (p<0.01 vs CTL), Nox4 (p<0.01 vs CTL), p22phox (p<0.01 vs CTL), p47phox (p<0.001 vs CTL), Atf4 (p<0.05 vs CTL) and eIF2α (p<0.05 vs CTL), and protein expression of NOX2 (p<0.01 vs CTL), eIF2α (p<0.05 vs CTL) and PERK (p<0.001 vs CTL). Ang II also induced increased protein expression of Na+ exchangers NHE1 (p<0.01 vs CTL) and NHE3 (p<0.05 vs CTL), co‐transporters NKCC2 (p<0.001 vs CTL) and NCC (p<0.01 vs CTL), also Na+ epithelial channel ENaC α isoform in the medulla (p<0.001 vs CTL) and β isoform in cortex (p<0.01 vs CTL). Furthermore, the all stimulatory effects of Ang II were decreased by co‐treatment with losartan.

Conclusion

our results suggest that the chronic infusion of Ang II was able to stimulate the expression of mRNA to factors associated with ER stress and oxidative stress which may be related with increased expression of NHE1, NHE3, NKCC2, NCC and ENaC (α and β subunits).

Support or Funding Information

CNPq (140141/2016‐2), CAPES, FAPESP.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of neutral amino acid transporter LAT4 in mouse epithelia
Authors: Rajendran Anuradha , Poncet Nadège , Oparija Lalita , Herzog Brigitte , Verrey François
Published: June 2021

Amino acids (AAs) are not only building blocks of proteins, but also play key roles in energy metabolism and as (precursors of) neurotransmitters. To maintain amino acid homeostasis, small intestine (SI) and kidney play a major role by absorbing amino acids from food and preventing their loss in the urine, respectively. Given their zwitterionic nature, neutral AAs require transport proteins to cross cell membranes. LAT4 (Slc43a2), a facilitated diffusion uniporter specific for essential neutral amino acids (Leu, Ile, Val, Met and Phe) was first identified by Bodoy et al., 2005 and then shown by us to be highly expressed in the basolateral membranes of SI and kidney tubule epithelia. LAT4 global knockout caused a postnatal malnutrition‐like condition leading to death within 10 days, which was hypothesized to be due to a severe defect in intestinal absorption of AAs (Guetg et al. 2015). To test this hypothesis, we generated an intestine‐specific LAT4 knockout mouse model using Cre‐LoxP system. Intestine specific LAT4 knockout pups were viable and did not show any significant changes in body weight gain as compared to their control littermates even up to 2 months after birth. Quantitative PCR, western blot and immunofluorescence data showed a nearly 95% efficient knockout of LAT4 throughout the SI (n=4). However, these mice displayed no visible phenotype. Amino acid transport along the SI was then assessed by gavaging an AA mix containing radiolabelled LAT4 substrate 3H‐Leu. Blood samples were collected from the tail vein 10′ before and 2′,5′, 10′, 20′ and 60′ after gavage. Mice were then euthanized, SI divided into 4 equal parts and the intestinal tissue and its contents collected separately. Measuring the distribution of 3H‐Leu using a β‐counter revealed an accumulation of Leu in the initial segment of the SI of knockout mice (n=5, P<0.001 by ANOVA), accompanied by a delayed bolus movement. Thus, we show that LAT4 knockout in SI is not the sole or major reason for the lethal phenotype of LAT4 global knockout pups, although it could have contributed to the lethality. In parallel, we have generated a doxycycline‐inducible kidney tubule‐specific LAT4 knockout mouse model to assess the effects of LAT4 deletion in kidney, both in young and adult animals. Results obtained with this new model will allow us to determine whether the urinary loss of amino acids may have strongly contributed to the lethal phenotype of LAT4 global knockout mice or whether it was caused by the defect of LAT4 in other tissues.

Support or Funding Information

This project is funded by a grant of the Swiss National Foundation (SNF) to FV.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Claudin‐2 Confers Calcium Permeability to the Jejunum and Ileum in Early Life
Authors: Beggs Megan R , Plain Allen , Lee Justin J , Alexander R. Todd
Published: June 2021

Infants and children must maintain a net positive calcium (Ca2+) balance in order to achieve an optimal peak bone mineral density by early adulthood. Across the small intestine, Ca2+ is absorbed via transcellular and paracellular pathways although the molecular details of these pathways in early life are not well delineated. Claudins are tight junction proteins that confer selective permeability to epithelia. Claudins‐2, −12 and −15 are expressed and have been implicated in paracellular Ca2+ permeability across intestinal epithelia. To date, the functional contribution of these claudins to the Ca2+ permeability (PCa2+) of small intestine segments has not been defined. The objective of this study was therefore to 1) determine if PCa2+ across small intestine segments changed with postnatal development 2) to asses whether claudin‐2 or −12 contribute PCa2+ to the small intestine. To this end, basolateral to apical 45Ca2+ fluxes on ex vivo intestinal segments in Ussing chambers were employed to indirectly assess permeability to Ca2+. We observed greater 45Ca2+ flux in P14 vs 2‐month mice across the duodenum (31.8 ± 2.2 vs 9.5 ± 2.0 nmol/h/cm2, P<0.0001) and jejunum (42.3 ± 3.9 vs 21.8 ± 3.5 nmol/h/cm2, P<0.01) but not ileum (40.5 ± 3.2 vs 30.1 ± 5.7 nmol/h/cm2, P=0.13). Next, we measured PCa2+ directly in Ussing chambers using diffusion potentials. PCa2+ was significantly greater in younger mice across the duodenum (1.43 ± 0.04 vs 1.13 ± 0.08 × 10−4cm/s, P<0.01), jejunum (1.64 ± 0.09 vs 0.84 ± 0.04 × 10−4cm/s, P<0.0001) and ileum (1.72 ± 0.08 vs 0.81 ± 0.05 × 10−4cm/s, P<0.0001). To determine if claudin‐2 or −12 facilitates greater PCa2+ at P14, diffusion potential experiments were repeated on Cldn2 or Cldn12 knockout (KO) mice. PCa2+ was decreased in Cldn2 KO mice at P14 across the jejunum (1.85 ± 0.09 vs 1.24 ± 0.07 × 10−4cm/s, P<0.0001) and ileum (1.92 ± 0.07 vs 1.09 ± 0.06 × 10−4cm/s, P<0.0001) to levels not different than 2‐month WT and KO mice. No differences were observed between Cldn12 knockout mice compared to wildtype littermates at either age. We conclude that the younger mice have enhanced Ca2+ permeability along the small intestine relative to older animals. Further, the increased jejunal and ileal Ca2+ permeability is mediated by claudin‐2 which likely contributes to a positive calcium balance for normal growth.

Support or Funding Information

This work was funded by grants from the Women and Children's Health Research Institute, which is supported by the Stollery Children's Hospital Foundation, and the National Sciences and Engineering Research Council to RTA, who is the Canada Research Chair in Renal Epithelial Transport Physiology. MRB is supported by a Vanier Canada Graduate Scholarship, and Alberta Innovates Clinician Fellowship.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
H89 Phosphoproteomics in PKA‐Null Cells
Authors: Kelleher Andrew , Limbutara Kavee , Yang Chin‐Rang , Raghuram Viswanathan , Knepper Mark
Published: June 2021

Protein phosphorylation, mediated by protein kinases, plays a crucial role in cellular regulation. One of the most important protein kinases is protein kinase A (PKA). N‐[2‐p‐bromocinnamylaminoethyl]‐5‐isoquinolinesulphonamide (H89) is often used as a “PKA specific inhibitor” to study the involvement of PKA in signaling pathways. However, evidence from cell‐free experiments has suggested that H89 can also inhibit other protein kinases. In this study, previously generated PKA‐null and PKA‐intact mouse cell lines were treated with H89 over a range of concentrations commonly used in the literature, followed by mass spectrometry‐based phosphoproteomics to globally assess changes in phosphorylation. From a total of 14,507 phosphorylation sites quantified, we found that 578 and 267 phosphorylation sites with significant changes in abundance in PKA‐intact and PKA‐null cells, respectively. Analyses of sequence logos generated from significantly decreased phosphorylation sites in PKA‐intact and PKA‐null cells both revealed a preference for basic amino acids at position −3 and −2. Thus, H89 appears to inhibit basophilic kinases, even in the absence of PKA. We conclude that, in intact cells, H89 can affect activities of protein kinases other than PKA, and therefore responses to H89 should not be regarded as a sufficient evidence for PKA involvement in a signaling process.

Support or Funding Information

This work was funded by the Division of Intramural Research, National Heart, Lung, and Blood Institute (projects ZIA‐HL001285 and ZIA‐HL006129, MAK). Andrew Kelleher was a member of the Biomedical Engineering Student Internship Program (BESIP, Robert Lutz, Director) supported by the National Institute for Biomedical Imaging and Bioengineering (June–August 2018). All mass spectrometry measurements were performed by the NHLBI Proteomics Core Facility (M. Gucek, Director).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Systemic Maternal Inflammation and Postnatal Hyperoxia Induces Early Life Cardiovascular Abnormalities in Mice
Authors: Pavlek Leeann R , Rogers Lynette
Published: June 2021

Background

Premature infants have a higher incidence of heart failure, cardiac hypertrophy, and ventricular systolic dysfunction as young adults. The mechanism underlying these abnormalities likely include changes in proteins related to the structure and function of the heart including connexin 43, α myosin heavy chain (αMHC), and β myosin heavy chain (βMHC). microRNAs, including miR‐208a and miR‐208b, play a role in the regulation of myosin heavy chain expression. In adult mice, abnormalities in these proteins and microRNAs are triggered by perinatal inflammation and postnatal hyperoxia. These investigations test our hypothesis that systemic maternal inflammation and neonatal hyperoxia induce changes in the structural and conduction proteins of the mouse heart and are present in embryonic and newborn mice.

Methods

Pregnant C3H/HeN mice were injected on embryonic day 16 (E16) with intraperitoneal LPS (80 μg/kg) to induce perinatal inflammation or saline. Offspring were euthanized on E18 or day of life 1. Another group of offspring were placed into room air (RA) or 85% O2 for 7 days after birth prior to euthanizing. Total RNAs were isolated from frozen whole heart samples. RT‐PCR was used to examine mRNA expression of connexin 43, αMHC, and βMHC. RT‐PCR was also used to assess expression of miR‐208a and miR‐208b. Data were analyzed using an unpaired t‐test.

Results

Maternal LPS induces decreases in miR‐208b that are present at E18 (10‐fold, P<0.05) and persist at day of life 1 (15‐fold, P<0.05). At day of life 7, oxygen exposure induces a downregulation of miR‐208b (1.8‐fold, P<0.05) compared to room air. The inflammation‐exposed mice have significant decreases in miR‐208a (9‐fold, P<0.05) and αMHC mRNA (30,000‐fold, P<0.05) on day of life 1. By day of life 7, mice exposed to the combination of inflammation and hyperoxia have less (1.6‐fold, P<0.05) connexin 43 mRNA compared to the control mice; no changes in connexin 43 are seen at day of life 1 or at day of life 7 with exposure to inflammation without hyperoxia.

Conclusions

Mice exposed to perinatal inflammation and postnatal hyperoxia demonstrate changes in structural and conduction components of the heart that are evident at the time of birth.

Support or Funding Information

NIH NICHD R01HD088033

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CC chemokine ligand 2 (CCL2) stimulates local aromatase expression in mammary adipose tissue via MAPK/ERK signaling pathway
Authors: Martínez‐Chacón Gabriela , Saarinen Niina , Mäkelä Sari
Published: June 2021

Obesity is an important risk factor for the most common type of breast cancer, i.e., estrogen‐dependent postmenopausal breast cancer. Obesity‐related inflammation, defined by enhanced tissue macrophage infiltration and increased levels of proinflammatory factors, stimulates estrogen production and, in turn, promotes tumor formation and progression. The augmented inflammatory milieu activating aromatase expression, the rate‐limiting enzyme for estrogen synthesis, is suggested to be the major mechanism behind the increased risk for breast cancer. The regulation of aromatase (CYP19A1) gene expression in breast tissue is complex and not fully understood. This study focuses on the role of CCL2, a macrophage recruiter often overexpressed in the obese and cancerous breast tissue, on the aromatase expression in mammary adipose tissue. We utilized mammary adipose tissue explants obtained from female hARO‐Luc aromatase reporter mice and cultured primary human breast adipose stromal cells (hASCs). In tissue explants, aromatase expression was measured by quantification of luciferase reporter activity, while in cultured hASCs, qPCR was used to examine expression of CYP19A1 and its specific promoter I.4 and II transcripts. Both, in mammary adipose tissue and in hASCs, CCL2 induced aromatase expression via the distal promoter I.4. Moreover, inhibitors of CCL2 receptor and MAPKs pathway, ERK1/2, blocked CCL2–induced CYP19A1 and PI.4 mRNA expression. In conclusion, these findings indicate that CCL2, an intrinsic pro‐inflammatory chemokine in adipose tissue, may stimulate aromatase expression and play an important role in controlling estrogen biosynthesis in the breast adipose stroma.

Support or Funding Information

This research is supported by the University of Turku Graduate School, the Finnish Cultural Foundation, the Turun yliopistosäätiö and the Institute of Biomedicine, University of Turku

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Single Prolonged Stress as a Prospective Model for Posttraumatic Stress Disorder in Females
Authors: Nwokafor Chiso , Navhi Roxanna J , Serova Lidia , Sabban Esther
Published: June 2021

Background

Sex plays an important role in susceptibility to stress triggered disorders. Posttraumatic Stress disorder (PTSD), a debilitating psychiatric disorder developed after exposure to a traumatic event, is two times more prevalent in women than men. However, the vast majority of animal models of PTSD, including single prolonged stress (SPS), were performed mostly with males.

Objectives

(1) Evaluate SPS as an appropriate model for PTSD in female rodents in terms of anxiety, depressive symptoms and changes of gene expression in the locus coeruleus; (2) Asses sex differences in SPS‐triggered changes in these measures; (3) Examine intranasal NPY as a potential early intervention for females.

Methods

Female rats were subjected to SPS and given either intranasal NPY or vehicle. Twelve and seven days later they were compared to unstressed controls on forced swim test (FST) and elevated plus maze (EPM) respectively. Changes in gene expression in the locus coeruleus (LC) were determined by quantitative RT‐PCR.

Results and Interpretation

SPS led to increased anxiety‐like behavior on EPM, and tended to raise depressive‐like behavior on FST. Following FST rats displayed elevated TH, CRHR1 and Y1R mRNA levels in the LC, consistent with increased activation of the noradrenergic system.

The expression level of these mRNAs were unchanged following EPM. Intranasal NPY at the doses shown to be effective in males, did not prevent development of depressive or anxiety‐like behavior or molecular changes in the LC. SPS may be milder for females when compared to males, as they did not show despair or immobility as readily as males on the forced swim component of the SPS protocols. In addition, after SPS stressors females exhibited less immobility on FST than males, however there were little overall sex differences in anxiety‐like behavior on EPM.

Conclusions

The results indicate that while SPS could be an appropriate PTSD model for females, there are important sex differences, such as response to NPY, that need to be taken into account.

Support or Funding Information

This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the DOD Department of Defense Broad Agency Announcement for Extramural Medical Research under Award No W81XWH‐16‐1‐0016 and by funds from the NYMC/Touro Bridge Funding Program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelin B Receptor Blockade Lowers Fasting Blood Glucose and Improves Insulin Sensitivity in Rats
Authors: Williams London J , Jenkins Haley N , Speed Joshua S
Published: June 2021

Elevated endothelin‐1 (ET‐1) is associated with obesity, as well as insulin resistance. We previously reported that rats lacking functional endothelin type B receptors (ETB) demonstrate improved glucose control. In addition, ETB receptor deficient rats have hypertension due to overactivation of the endothelin type A receptor (ETA). In order to determine the effects of ETA receptor activation on insulin sensitivity, we tested the hypothesis that pharmacological blockade of ETB receptors and dual ETA/ETB receptor inhibition would improve glucose control in Sprague Dawley (SD) rats. SD rats were treated with Bosentan (30 mg/kg/day), Atrasentan (10 mg/kg/day), A‐192621 (10 mg/kg/day), or vehicle over a period of two weeks. At day 7, insulin tolerance testing was performed and tissues were collected at day 14 after a 6‐hour fasting period. Treatment with either the dual ETA/ETB antagonist Bosentan, or the ETB antagonist A‐192621, significantly lowered fasting blood glucose as compared to either vehicle or ETA antagonist (Atrasentan) (82.72±4.2*, 75±6.1*, 99.1±2.0, and 94.8±3.2 mg/dl respectively, *p<0.05 vs. vehicle). In addition, both Bosentan and A‐192621 improved insulin tolerance compared to vehicle treated, while Atrasentan had no effect. There were no differences in plasma cholesterol (LDL or HDL) or triglycerides. These data indicate that blockade of ETB receptors improves glucose control and may be an effective treatment for insulin resistance and pre‐diabetes.

Support or Funding Information

This work is supported by NHLBI grant R00 HL127178 to JSS.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.