bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced vestibular function in veterans is associated with worse cerebral autoregulation (1068.15)
Authors: Serrador Jorge , Acosta Amanda , Ghobreal Bemin , Blatt Melissa
Published: June 2021

Recent data has demonstrated that veterans returning from Iraq and Afghanistan have increased incidence of mild traumatic brain injury (mTBI) as well as impaired vestibular function. The objective of this work is to determine if reduced vestibular function is associated with impaired cerebral autoregulation. To asses vestibular function we measured ocular torsion, rotation of the eye within the socket during roll tilt. This vestibular ocular reflex provides us an index of otolith function. To assess cerebral autoregulation we had veterans perform three sit to stand maneuvers while measuring beat‐by‐beat blood pressure, cerebral flow velocity in the middle (MCA) and anterior cerebral artery (ACA) and end tidal CO2. 11 Veterans completed both vestibular and cerebral autoregulation assessments. Ocular torsion ranged from 0.03 to 0.32 deg torsion per deg head tilt (Mean 0.14). Ocular torsion was correlated to autoregulatory index (ARI) in the MCA (R2=0.115, P=0.058) and the ACA (R2=0.212, P=0.018), with lower ocular torsion having reduced ARI. Defining low ocular torsion using a mean split, we found significantly lower ARI values in both MCA (Low 3.4±0.3 vs High 4.7±0.5, P=0.014) and ACA (3.3±0.3 vs 5.0±0.5, P=0.003). These data suggest that impaired vestibular function in these veterans is associated with worse cerebral autoregulation. One possible explanation is that veterans with reduced torsion also have mTBI and thus both the reduced vestibular function and the impaired autoregulation are a consequence of mTBI. Further work is needed to examine the connection between mTBI, vestibular function and cerebral autoregulation.

Grant Funding Source: Supported by Dept of Veteran Affairs and NIH R21 DC009900
Lack of a sex difference in leptin‐mediated regulation of appetite, cardiovascular function and glucose homeostasis (1083.5)
Authors: da Silva Alexandre , Pinkerton Mark , Spradley Frank , Palei Ana , do Carmo Jussara
Published: June 2021

Previous studies using male rodents have shown that the adipocyte‐derived hormone leptin acts in the brain to regulate cardiovascular function and energy homeostasis noted by modulation of appetite and energy expenditure and normalization of diabetes‐induced elevations in glucose levels. Because it is uncertain if leptin acts similarly in females, the aim of our study was to examine sex differences in leptin’s actions on blood pressure (BP), heart rate (HR), appetite, and glucose homeostasis. Female Sprague‐Dawley rats (n=6) and male controls (n=5) were instrumented with intracerebroventricular (ICV) cannulas in the lateral ventricle for continuous 7‐day infusion of leptin (15 μg/day) and telemetry probes for measurement of BP and HR 24‐hr/day. At baseline, female rats had significantly lower MAP (96±3 vs. 104±4 mmHg, p<0.05) but higher HR (375±5 vs. 335±5 bpm, p<0.05) compared to males but, following leptin treatment, we observed similar rises in BP (~3 mmHg) and HR (~25 bpm) in both sexes. Females had significantly reduced body weight (BW, 283±2 vs. 417±7 g, p<0.05) and lower caloric intake (162±20 vs. 192±9 kcal/kg of BW, p<0.05) compared to males, and leptin infusion reduced BW (‐10%) and caloric intake (‐62%) in both sexes alike. Also, in females and males, leptin infusion significantly reduced, to a similar extent, both fasting insulin (1.9±0.5 vs. 0.6±0.3 and 1.9±0.2 vs. 0.3±0.1 ng/ml, respectively) and blood glucose levels (100±6 vs. 75±6 and 80±4 vs. 73±5 mg/dl, respectively). In female rats with streptozotocin‐induced diabetes (n=5), chronic ICV leptin treatment for 7‐days also completely normalized the elevated glucose levels (315±33 vs. 83±13 mg/dl, p<0.05). These results show that the effects of leptin on cardiovascular function, glucose regulation and energy homeostasis are similar in male and female rats. Therefore, our results provide no evidence of a sex difference in leptin’s cardiovascular or metabolic actions. (NHLBI‐PO1HL51971, NIGMS P20GM104357, AHA‐SDG5680016)

Grant Funding Source: NIH: NHLBI‐PO1HL51971 and NIGMS P20GM104357; AHA: SDG5680016
Role of Src homology 2 domain‐containing protein B in the regulation of GFR and renal afferent arteriole responsiveness (1088.12)
Authors: Gao Xiang , Sällström Johan , Ma Zufu , Welsh Michael , Persson A. Erik
Published: June 2021

BACKGROUND: Src homology 2 domain‐containing protein B (Shb) is an adapter protein which regulates several signal transduction cascades and endothelial cell functions. We used Shb‐knockout (Shb‐/‐) and wild‐type (Shb+/+) mice to investigate the role of Shb in regulating glomerular filtration rate (GFR), vascular resistance and tubuloglomerular feedback.

METHODS: GFR was measured in conscious Shb‐/‐ and Shb+/+ mice using FITC‐inulin. Isotonic contractions were measured in isolated and perfused renal afferent arterioles from Shb‐/‐ and Shb+/+ mice. Concentration responses to Ang II (10‐12 to 10‐6M; 2 minutes each) doses, low‐dose Ado (10‐8 mol/l; 15 min) alone or Nω‐nitro‐l‐arginine methyl ester (L‐NAME; 10‐4 mol/l; 15 min) alone, as well as Ado (10‐8 mol/l) or nitric oxide (NO) synthase inhibitor L‐NAME (10‐4 mol/l) in combination with cumulative application of Ang II (10‐12 to 10‐6 mol/l; 2 minutes each) were studied in both genotypes.

RESULTS: There was a significantly increased GFR (371 ± 12 µl/min, n=11) in Shb‐/‐ comparing to Shb+/+ (321 ± 11 µl/min, n=8) mice. The maximal arteriolar contraction to Ang II was remarkably larger in Shb‐/‐ (87 %, n=8) than in Shb+/+ (54 %, n=8) mice. Low‐dose Ado contracted afferent arterioles in both genotypes (6% in Shb‐/‐ and 7% in Shb+/+). Ado significantly enhanced Ang II constriction in afferent arterioles in both genotypes (to 93% in Shb‐/‐ and to 72 % in Shb+/+). L‐NAME reduced arteriolar diameters significantly more in Shb‐/‐ (14 %, n=7) than in wild types (9 %, n=7) and remarkably augmented ANG II responses in both genotypes. L‐NAME with Ang II almost fully constricted renal afferent arterioles in Shb‐/‐ (98 %), while contracted 64% in Shb+/+ mice.

CONCLUSION: The absence of Shb markedly increases GFR. Both low‐dose adenosine and L‐NAME treatments significantly augment Ang II arteriolar constriction effectiveness, which indicates Ado‐Ang II and NO‐Ang II interactions in both Shb‐/‐ and Shb+/+ mice. The underlying mechanisms remain to be resolved.
Nucleic acid binding of annexin A2 is regulated through angiotensin II/AT1 signaling in kidneys of hypertensive rats (1088.2)
Authors: Hultström Michael , Paliege Alexander , Skogstrand Trude , Fähling Michael
Published: June 2021

The aim was to identify new targets that regulate gene expression at the post‐transcriptional level in hypertension. Heparin affinity chromatography was applied to enrich putative nucleic acid‐binding proteins from kidneys of 2K1C hypertensive rats. A 0.2mm silver clip was used to induce hypertension (sham: 109±8 mmHg, 2K1C: 146±6 mmHg, P<0.05). We found a 36 kDa protein that showed an increased affinity to the nucleic acid mimetic in the 2K1C model. This factor was identified by LCMSMS and western blotting as Annexin A2 (ANXA2), a RNA‐binding protein known to influence mRNA stability and translation. Immunofluorescence staining revealed widespread distribution of ANXA2 throughout the kidney with high levels in endothelial cells, the cells of the Bowman capsule and in the medullary collecting ducts. Infusion of angiotensin II (AngII) reproduced our findings in the 2K1C animals and the AT1a blocker Losartan abolished the effect on Annexin A2. Strikingly, we observed no change in total Annexin A2 protein levels or localization, suggesting that post‐translational modification modulates Annexin A2 function via ANGII/AT1a signalling. In conclusion, AngII regulates the nucleic acid binding activity of Annexin A2 in the kidneys of rats with hypertension and, thus, may be an important regulator of gene expression in this setting.

Grant Funding Source: Supported by Lars Hierta Foundation, Magnus Bergvall Foundation, Swedish Society for Medical Researc
G protein‐coupled receptor expression and function in pulmonary artery smooth muscle cells: new targets in pulmonary arterial hypertension (1090.3)
Authors: McDonald Daniel , Aroonsakool Nakon , Kwon Ohmin , Insel Paul , Murray Fiona
Published: June 2021

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance, in part due to increased proliferation of pulmonary artery smooth muscle cells (PASMC). Since 3’5’‐cyclic adenosine monophosphate (cAMP) decreases proliferation of PASMC, G protein‐coupled receptors (GPCRs) that couple to Gαs or Gαi are attractive targets for the treatment of PAH. We used a real‐time PCR GPCR array to identify and quantify the GPCRs expressed by PASMC isolated from normal subjects and from patients with PAH. We found that human PASMC express >135 GPCRs, at least 60 of which regulate cAMP formation. GPCR expression correlates with function: for Gαs‐coupled GPCRs with formation of cAMP and inhibition of cell proliferation, thus documenting that the GPCR array identifies physiologically relevant GPCRs. PAH‐PASMC (both from idiopathic and secondary PAH patients) have an increase (>2‐fold) in the expression of 41 GPCRs compared to control‐PASMC, including multiple GPCRs that link to Gαs or Gαi. In addition, PAH‐PASMC have a higher expression of GPCRs that preferentially couple to Gαq/11 or to Gα12/13. Taken together these data provide evidence that a GPCRomic approach can identify GPCRs that may contribute to the physiology of PASMC and that may be new druggable targets for PAH, a disease for which no currently optimal therapies exist.

Grant Funding Source: NIH
Overexpression of HSP72 protects against mechanical ventilation‐induced mitochondrial dysfunction and apoptotic signaling in the diaphragm (1091.1)
Authors: Smuder Ashley , Sollanek Kurt , Min Kisuk , Kwon Oh Sung , Nelson W , Powers Scott
Published: June 2021

Mechanical ventilation (MV) is a life‐saving measure for patients in respiratory failure. However, prolonged MV results in diaphragm weakness, which contributes to problems in weaning from the ventilator. Therefore, developing effective countermeasures to protect against MV‐induced diaphragm weakness is important. In this regard, HSP72 overexpression has been demonstrated to protect against skeletal muscle dysfunction during a variety of conditions (i.e. hindlimb immobilization and cancer cachexia). Therefore, the current study tested the hypothesis that HSP72 overexpression in the diaphragm would protect against MV‐induced diaphragm dysfunction. Cause and effect was determined using a novel technique to overexpress HSP72 in the rat diaphragm via an adeno‐associated virus vector delivered directly to the diaphragm. Our results demonstrate that 12 hours of MV results in impaired mitochondrial function and a significant increase in mitochondrial ROS release, which leads to caspase‐3 activation and increased apoptosis within the diaphragm. Overexpression of HSP72 in the diaphragm protected against these alterations and inhibited MV‐induced apoptotic signaling. Therefore, HSP72 overexpression in the diaphragm may have therapeutic potential to combat MV‐induced diaphragm weakness.

Grant Funding Source: This work was supported by NIH R01 AR064189 awarded to SK Powers.
Novel signaling function of hCLCA1 in airway macrophages activation (1095.2)
Authors: Ching John , Loewen Matthew
Published: June 2021

The CLCA gene family produces both secreted and membrane‐associated proteins that modulate ion‐channel function, drive mucus production and have a poorly understood pleiotropic effect on airway inflammation. In airway inflammation, the primary up‐regulated human CLCA ortholog is hCLCA1. Here we show that secreted hCLCA1 is able to activate macrophages, inducing them to express cytokines and to undertake a pivotal role in airway inflammation. Conditioned media collected from HEK293 cells heterologously expressing hCLCA1 increased the expression of pro‐inflammatory cytokines (IL‐1β, IL‐6, TNF‐α, and IL‐8) in U‐937 monocyte‐macrophage cell line. Similar pro‐inflammatory response was achieved in primary porcine alveolar macrophages activated with hCLCA1 conditioned media, showing that the effect was not cell line dependent. Immuno‐purified hCLCA1 at physiologically relevant concentration of ~100 pg/mL was also able to elicit pro‐inflammatory response in macrophages. These findings demonstrating that secreted hCLCA1 is able to function as a signaling molecule and to regulate cytokine expression in macrophages.

Grant Funding Source: NSERC
Characterization of the immune response in male and female mice following lipopolysaccharide‐induced systemic inflammation (1096.10)
Authors: Bubalo Nina , Nguyen Peter , Nguyen Tuan , Abramson Tzvia , Wilkinson Katherine
Published: June 2021

Intraperitoneal injection of lipopolysaccharide (LPS) is a common method to induce systemic inflammation, but there is wide variability in the doses given and no standardized measure of inflammatory level. We characterized the inflammatory response in adult male and female C57BL/6 mice following 3 LPS exposure protocols by measuring body weight changes, spleen weight and immune cell composition in the blood using flow cytometry. Mice were injected twice a week for 1 month with either a 200 μL dose of saline or a low dose of LPS (7.5 x 10^4 EU/kg). Half the animals in each condition were injected with a high dose of LPS (7.5 X 10^5 EU/kg) the day before sacrifice. Acute exposure to a high dose of LPS caused a 10.6% decrease in body weight overnight and no significant change in spleen weight. White blood cell and lymphocyte counts decreased and toll‐like receptor 4 (TLR4) positive macrophage count increased for both males and females. Exposure to 1 month of low LPS caused no change in the immune cell composition in the blood and body weight, but spleen weights were significantly increased compared to saline animals. 1 month of low LPS followed by high LPS caused an even greater increase in spleen weight than the 1 month low LPS condition, but no other changes. In summary, all 3 LPS exposure protocols led to evidence of an immune response in both sexes, but body weight and blood immune cell composition normalized following chronic low LPS exposure.

Grant Funding Source: Supported by a CSUPERB New Investigator Award (KAW)
The alkaline anterior midgut of larval mosquitoes as a barrier for microorganisms (1100.5)
Authors: Onken Horst , Bassous Monica , Moffett David , Corbo Christopher
Published: June 2021

The lumen of the anterior midgut of larval mosquitoes is maintained at extremely alkaline pH values of 10‐12, and digestive enzymes show very alkaline pH optima. This situation favors digestion of denatured protein like in acidic mammalian stomachs. In the present study, we are evaluating if the extremely alkaline pH region in the anterior midgut of 4th instar larvae of yellow fever mosquitoes (Aedes aegypti) also serves as a barrier for microorganisms. Bacteria were identified with conventional, microbiological techniques by utilizing a series of biochemical assays. Samples involved the water (with food), unsterilized and sterilized larvae, as well as head/thorax and abdomen of sterilized larvae. The fifteen different microorganisms identified included species of gram‐negative bacilli, especially gammaproteobacteria, and a few gram‐positive rods. The presence of different species of bacteria in the anterior (head/thorax) and posterior (abdomen) parts of the animals indicates that the alkaline region in the midgut of larval Aedes aegypti does indeed serve as a selective barrier for bacteria.
Effects of muscle specific VEGF loss on muscle hypertrophy and function induced by functional overload in mice (1102.6)
Authors: Huey Kimberly , Delavar Hamid , Breen Ellen
Published: June 2021

Functional overload (FO) stimulates muscle growth and increases strength and endurance. Vascular endothelial growth factor (VEGF) is essential for exercise‐induced angiogenesis. We hypothesized VEGF expressed by skeletal myofibers is critical for muscle growth and improvements in force production and fatigue resistance. To test this hypothesis, adult mice with conditional skeletal myofiber specific VEGF gene deletion (skmVEGF‐/‐) and wild type (WT) littermates underwent functional overload (FO) of the plantaris (removal of gastrocnemius and soleus) or sham surgery. In vivo plantaris isometric force and fatigue resistance (% of maximal force after 10 contractions) were measured 7 or 14d after FO or sham surgery with a dual mode foot plate system in anesthetized skmVEGF‐/‐ or WT mice. Mass increased ~ 35% in both 14d‐FO groups compared to sham (p<0.01). In WT mice subjected to FO, maximal isometric force was unchanged at 7d and increased at 14d (sham, 0.37±0.03; FO, 0.46±0.04 g/mg body mass, p<0.05). In skmVEGF‐/‐ mice, FO was associated with a decrease in maximal force after 7d (sham, 0.39±0.0; FO, 0.23±0.03 g/mg body mass, p<0.05) and 14d (sham, 0.39±0.03; FO, 0.27±0.04 g/mg body mass, p<0.05). Fatigue resistance was not different between groups at 7d. After 14d of FO, fatigue resistance increased from 58±3%‐sham to 73±2%‐FO of maximal force in WT mice (p<0.05) and was unchanged in skmVEGF‐/‐ mice. These data suggest that VEGF‐dependent functions are essential for improving muscle strength and fatigue resistance, independent of muscle growth, in response to a hypertrophic stimulus.

Grant Funding Source: Supported by NIH R15AR060469
Radiation combined with hind‐limb suspension affects physiological parameters in rats (1103.7)
Authors: Chowdhury Parimal , Long Ashley , Akel Nisreen , Gaddy Dana , Griffin Robert , Bailey Lauren , Dobretsov Maxim
Published: June 2021

This study examines acute and degenerative tissue responses to space‐like radiation doses in a rodent model of simulated microgravity. Three groups of rats, control (CON), irradiated (IR) and irradiated plus suspended (IR+HLS) were maintained for two weeks. IR and IR+HLS groups were exposed to radiation dose of 10GY. Body weights, soleus muscle weights, malondialdehyde (MDA), insulin/glucose levels, pressure‐pain threshold (PPT), grip force and heat pain were measured. CON gained weight faster than IR or IR+HLS. HLS but not IR resulted in soleus muscle atrophy. Bone mineral density in the proximal tibia was compromised by HLS and decreased by IR+HLS. Random plasma glucose levels were not affected by IR or IR+HLS. IR increased plasma insulin levels. PPT was increased in CON, remains unchanged in IR and decreased in (IR+HLS). MDA in plasma or in soleus muscle remain almost unchanged in CON, IR and (IR HLS) rats. However, IR significantly increased soleus muscle Cyt / Arg ratio over CON. Grip force, and/or low back pain did not differ between CON, IR and IR+HLS groups. No effects of irradiation were seen in heat pain threshold measurements. IR eliminated “hypoalgesia” and (IR+HLS) produced hyperalgesia. Moreover, IR exacerbated the HLS‐induced bone loss in the proximal tibia. These data suggest that radiation induces major physiological changes and these changes are exacerbated by exposure to HLS.

Grant Funding Source: Arkansas Space Grant Consortium
Effect of passive heating on arterial compliance in smokers vs. non‐smokers (1106.1)
Authors: Moyen Nicole , Ganio Matthew , Burchfield Jenna , Tucker Matthew , Gonzalez Melina , Dougherty Erin , Robinson Forrest , Ridings Christian , Veilleux Jennifer
Published: June 2021

An inverse relationship exists between baseline arterial compliance (AC) and changes with passive heating in non‐smokers, yet it is unknown if this relationship exists in smokers. Purpose: To examine the effects of passive heating on AC in chronic smokers (S) vs. non‐smokers (NonS). Methods: 13 S and 13 NonS (27.2 ± 7.9 y; 78.8 ± 15.4 kg; 177.6 ± 6.7 cm) were passively heated until core temperature (TC) increased 1.5 °C from baseline. At baseline and each 0.5 °C TC increase, AC was assessed peripherally and centrally using an ultrasound measuring pulse wave velocity (pPWV and cPWV, respectively). Results: Independent of group, cPWV significantly increased (p < 0.005) from baseline (grand mean: 526.7 ± 81.7 cm/s) to 1.5 °C (579.7 ± 69.8 cm/s). Regardless of group, pPWV did not change with heating (grand mean: baseline = 691.9 ± 92.9; 1.5 °C = 691.9 ± 79.5 cm/s; p > 0.05). Changes in cPWV and pPWV with heating negatively correlated (p < 0.05) with baseline PWV in S (cPWV: r = ‐0.59; pPWV: r = ‐0.62) and NonS (cPWV: r = ‐0.45; pPWV: r = ‐0.77). However, the magnitude of these relationships did not differ between S and NonS, either centrally or peripherally (p > 0.05). Conclusion: Passive heating to a 1.5 °C TC increase similarly affected AC in S and NonS. Inverse relationships between baseline PWV and changes in PWV with heating suggest the effect of heat on AC depends on baseline normothermic compliance.

Grant Funding Source: Supported by: Arkansas Biosciences Institute, Arkansas Tobacco Settlement Proceeds Act of 2000
A role for the circadian clock protein Per1 in the regulation of NHE3 and SGLT1 in human proximal tubule cells (1109.4)
Authors: Richards Jacob , All Sean , Cheng Kit‐Yan , Gumz Michelle
Published: June 2021

Mounting evidence has demonstrated that renal function and blood pressure undergo circadian oscillations. Our lab has previously demonstrated that the circadian clock protein Per1 regulates multiple genes involved in sodium reabsorption in the renal collecting duct, but other parts of the nephron have not been investigated. The proximal tubule is responsible for a majority of sodium reabsorption. The goal of this study was to determine if NHE3 and SGLT1 were regulated by the circadian clock protein Per1. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of SGLT1 and NHE3, but not SGLT2 in the renal cortex of mice. These findings were confirmed using Per1 siRNA and pharmacological blockade of Per1 nuclear entry in HK‐2 human proximal tubule cells. Transcriptional regulation was demonstrated by measuring short‐lived hnRNA. Chromatin‐immunoprecipitation experiments demonstrated the interaction of Per1 and CLOCK with the promoters of NHE3 and SGLT1, an effect that was reduced by blockade of Per1 nuclear entry. Importantly, both membrane and intracellular protein levels of NHE3 and SGLT1 were decreased following blockade of nuclear Per1 entry. These data demonstrate a role for Per1 in the transcriptional regulation of NHE3 and SGLT1 in proximal tubule cells.

Grant Funding Source: Supported by NIH, ASN and AHA
An emerging link between the negative EGFR feedback regulator Mig6 and hepatic glucose production (1115.5)
Authors: Lutkewitte Andrew
Published: June 2021

Hepatic glucose production (HGP) from either glycogenolysis or de novo gluconeogenesis requires the hydrolytic cleavage of glucose‐6‐phosphate to glucose and inorganic phosphate by the glucose‐6‐phosphatase catalytic subunit (G6Pase). G6Pase is induced during fasting, type 2 diabetes (T2D), and endoplasmic reticulum (ER) stress to promote HGP. In contrast, G6Pase can be suppressed by hormonal signals including activation of the epidermal growth factor receptor (EGFR). As a negative regulator of EGFR, Mitogen‐inducible gene 6 (Mig6) could be a novel target for modulating HGP. Thus, we hypothesized that Mig6 induction increases HGP by inhibiting EGFR‐mediated suppression of G6Pase. Using C57Bl/6 mice and HepG2 cells, we determined that Mig6, like G6Pase, is induced during fasting, diabetes, and ER stress. To further study this regulation, we generated liver‐specific Mig6 knockout (LKO) mice by crossing Mig6flox/flox mice with albumin‐Cre transgenic mice. Compared to wild‐type littermates, LKO mice had a 2‐fold increase in liver weight and an increase in total liver glycogen. Moreover, LKO had reduced G6Pase expression with no change in glucokinase or PEPCK gene expression, suggesting that EGFR signaling restrains G6Pase expression in vivo. We conclude that similar to G6Pase, Mig6 is dynamically regulated during fasting, obesity‐induced diabetes, and ER stress, thus providing a new therapeutic target for unrestrained HGP in T2D.

Grant Funding Source: Supported by a Showalter Research Trust Award from IUSM
Pterostilbene, a natural analogue of resveratrol, decreases hepatic lipid deposition by increasing triacylglycerol hydrolases activity (1116.6)
Authors: Moreira Carolina , Rodrigues Angélica , Cordeiro Letícia , Mario Érica , Rosa Damiana , Botion Leida
Published: June 2021

In the present work the role of pterostilbene on lipid metabolism was evaluated in a rodent obesity model induced by monosodium glutamate (MSG) during the first ten days of life. At 70 days of age, control (C) and MSG (M) animals were divided into groups that received by gavage pterostilbene (P) (40 mg.kg‐1.day‐1) or vehicle (V) during 20 days. After removal of liver and adipose tissue, triacylglycerol (TAG) hydrolases activity was evaluated and SIRT‐1 (sirtuin‐1) expression was determined by real time PCR. The results obtained in fed rats showed increase in Lee index and visceral adiposity in MSG group (MV) in relation to control (CV). Treatment of obese rats with pterostilbene (MP) decreased Lee index with no change in adiposity as compared to MV. Serum TAG levels were increased in MV group (222±19 mg/dL) compared to CV (145±21 mg/dL) and MP group (212±19 mg/dL) did not show significant difference compared to MV. Fat liver deposition markedly increased in MV group when compared to CV, and decreased in MP group in relation to MV. Treatment with pterostilbene increased TAG‐hydrolytic activity in the liver of control and obese groups. In addition, SIRT‐1 mRNA expression increased in MP when compared to MV. Our data clearly established that the treatment of obese rats with pterostilbene has beneficial effects in the reduction of liver fat deposition by increasing TAG‐hydrolytic activity, probably by SIRT‐1 pathway.

Grant Funding Source: Supported by CNPq, CAPES, FAPEMIG
Drug‐induced constipation alters gut microbiota stability leading to physiological changes in the host (1118.2)
Authors: Touw Ketrija , Wang Yunwei , Leone Vanessa , Nadimpalli Anuradha , Nathaniel Hubert , Gianrico Farrugia , Kashyap Purna , Chang Eugene
Published: June 2021

Gut microbes play a significant role in development and maintenance of intestinal functions, but can contribute to pathogenesis of gastrointestinal (GI) disorders when host‐microbiome relationship is perturbed. In this study we sought to examine how constipation affects microbiome, and how these alterations in turn affect the host. Adult C57Bl/6 mice were treated with 0.1% Loperamide in drinking water for 7 days to induce constipation. Loperamide‐treated mice had increased GI transit time, indicating constipation. Bacterial DNA analysis by HiSeq sequencing showed decreased Firmicutes and increased Bacteroidetes in constipated mice. Microbiome metabolic potential was measured with tetrazolium dye assay and showed shift toward increased sugar metabolism in Loperamide‐treated mice. Microbial metabolites were measured by GC‐MS, and showed lower butyrate concentration in constipated mice. To determine constipation effects on the host, we introduced microbiota from constipated donors to germ‐free recipients. These mice had delayed GI transit time. To determine mechanism for delayed GI transit we analyzed colon tissues by qRT‐PCR and western blotting analysis, and determined lower expression of nNos mRNA and protein levels in these mice. Overall, these results suggest that delayed GI transit shifts microbiome taxonomic and metabolic profile, and these changes further contribute to development of constipation.

Grant Funding Source: NIH NIDDK T32 DK07074
Chronic ethanol intake leads to differential effects on the multivalent epigenetic state in liver (1120.1)
Authors: Patra Biswanath , Kuttippurathu Lakshmi , Hoek Jan , Vadigepalli Rajanikanth
Published: June 2021

The objective of this study was to characterize the effects of chronic alcohol consumption on the epigenetic state of key regulators of liver function, repair and regeneration. Rats were fed a liquid diet following established Lieber‐DeCarli protocol, with the treatment group of animals receiving 36% of total calories derived from ethanol, and pair‐fed control animals receiving an iso‐caloric liquid diet, for 5 weeks or 7 months.

Chromatin immunoprecipitation (ChIP) and high‐throughput RT‐PCR assays were performed to test the level of H3K27me3, H3K9me2, H3K9ac, H3K18ac at a selected target promoters, using Diagenode autoChIP protocols. Expression of H3 methyl and acetyl protein was tested by Western blotting. Overall histone acetylation and methylation levels were found to be decreased at both 5 week and 7 month time points as revealed by Western blot assays. However, nearly all of the histone markers tested showed differential methylation and acetylation, with significant increase at several key target promoters, at either 5 week or 7 month time frame of chronic ethanol intake. Our results reveal a combinatorial epigenetic state in which multiple histone modification markers were enriched at several target promoters, and the patterns of these multivalent states were altered in a complex manner during adaptation to chronic ethanol intake. Research Support: NIH R01 AA018873, K05 AA017261, and T32 AA007463.
The role of Toll‐like receptor 2 in macrophage activation (1125.3)
Authors: Jang Kyeong Ran , Gensel John
Published: June 2021

The role of Toll‐Like Receptor 2 (TLR2) in macrophage activation Kyeong Ran Jang1,2, John C. Gensel2, 1APS Undergraduate Summer Research Fellow; 2Spinal Cord and Brain Injury Research Center; Department of Physiology; University of Kentucky Macrophages (MO) are a hallmark of injury and can contribute to pathology or repair. In CNS trauma, MO induces axon dieback. Injecting TLR2 agonist to the site of CNS trauma reduces dieback, with increased MO activation. We hypothesize that TLR2 activation regulates the pathological potential of MO. TLR co‐activation with other stimuli induces a regulatory (M2b) MO phenotype. Through activation of TLR4, M2b MO releases IL10 (anti‐inflammatory cytokine) to help resolve inflammation. Little is known if TLR2 can drive an M2b phenotype. We tested our hypothesis by stimulating bone marrow‐derived and RAW cell MO with zymosan (ZYM). ZYM mimics MO activation at CNS trauma by co‐activation of TLR2 and other stimuli. 4 activation groups were tested: 1) untreated, 2) ZYM, 3) ZYM without TLR2 (DZYM), and 4) PAM2CSK4 (TLR2 agonist). All results are indicative of multiple independent replications. Examining IL10, IL12, CD80, CD86, and CCL1 gene markers after 6hrs of stimulation, ZYM stimulated MO significantly up‐regulated IL10 and CD86 (2 markers of the M2b MO). Notably, neither DZYM nor PAM2CSK4 alone was sufficient to drive M2b activation. There were no significant differences in other markers among groups and results were similar for both cell types. We conclude that TLR2 induces an M2b regulatory phenotype. Identifying TLR2 as target for MO opens new ways for therapeutic strategies to limit the pathological properties of MO.
Chronic intermittent hypoxia is associated with elevated pro‐opiomelanocortin mRNA and reduced microRNA‐375 levels in arcuate nucleus of hypothalamus (1126.5)
Authors: Nedungadi T. Prashant , Cunningham J. Thomas
Published: June 2021

Sleep apnea is a major risk to public health due to its close association with metabolic diseases. Exposure of rats to our 7 days of CIH protocol during their nocturnal period mimics repetitive bouts of arterial hypoxemia and elicits metabolic dysfunction characterized by reduced food intake (p<0.05, n=11), reduced body weight gain (p<0.005, n= 18), hyperinsulinemia (p<0.05, n=7) and glucose intolerance (p<0.05, n=8). However, CIH‐associated metabolic defects are not well understood because its onset is generally unknown. The arcuate nucleus of the hypothalamus (ARH) is a key hypothalamic site which integrates various metabolic cues to control energy homeostasis including anorexigenic POMC signalling. We have observed that the transcription factor FosB/ΔFosB, a well‐known marker of chronic neural transcriptional activation, is increased in ARH after CIH (p<0.05, n=6). POMC gene expression was significantly higher following CIH (P<0.05, n=4). Using miRNA PCR array (miScript‐ Qiagen), we screened for 84 miRNAs expressed in ARH punches that might be involved in energy regulation. In ARH, 29 of the 84 miRNAs tested were present in high abundance including miRNA 375, which has been previously linked as an important regulator of energy homeostasis and shown to have a negative regulatory relationship with POMC in the pituitary. Following CIH, miR‐375 was downregulated in ARH compared to normoxic controls (P<0.05, n=4). A possible association of miRNA‐375 with POMC may provide new insights concerning the regulation of POMC ARH neurons in CIH associated metabolic dysfunction.

Grant Funding Source: P01 HL88052
Effect of combined V1a/V1b vasopressin receptor blockade within the paraventricular nucleus on neurocardiovascular responses (1130.2)
Authors: Rossi Noreen , Li Cailian , Chen Haiping , Maliszewska‐Scislo Maria
Published: June 2021

The paraventricular nucleus (PVN) plays a critical role vasopressin (AVP) secretion by the neurohypophysis. AVP released from dendrites of magnocellular neurons within PVN influences systemic hemodynamics and renal sympathetic nerve activity (RSNA). Both V1a and V1b receptors exist on PVN neurons. We have shown that inhibition of either of these receptors alone blocks the rise in MAP and RSNA in response to AVP. We tested the hypothesis that combined subthreshold doses of V1a and V1b blockers within the PVN will inhibit AVP‐induced increases in MAP and RSNA. Studies were performed in awake, unrestrained,chronically instrmented male Sprague Dawley rats. Microinjection of 100 ng AVP into the PVN increased MAP 18.2±3.1 mmHg (P<0.001) while heart rate and RSNA decreased 52±21 bpm and 26.8±10.6%baseline, respectively (P<0.05). The response to combined subthreshold inhibitory doses of Manning compound (V1a, 20 ng) and nelivaptan (V1b, 0.5 ng) resulted in a significant attenuation of the rise in MAP, 13.3±3.2 mmHg, and attenuation of the reflex decrease in heart rate, 12±8 bpm, and RSNA, 7.2±4.6%baseline. Vehicle did not block any of the responses. These findings suggest that V1a and V1b activated pathways both participate and may interact to induce the pressor and sympathoexcitatory response to AVP within the PVN.

Grant Funding Source: VA Merit Award
Selective expression of an angiotensin‐(1‐7)‐producing fusion protein in the brain decreases blood pressure (1130.22)
Authors: Alves Daniele , Guimaraes Priscila , Todiras Mihail , Bader Michael , Santos Robson , Campagnole‐Santos Maria
Published: June 2021

Angiotensin‐(1‐7) [(Ang‐(1‐7)] attenuates arterial hypertension and improves baroreflex control upon chronic infusion in the central nervous system. In the present study we evaluated the baseline levels, variability and circadian pattern of blood pressure (BP) and heart rate (HR), as well as, the sensitivity of baroreflex control of heart rate and level of cardiac autonomic tone in transgenic rats that exhibit increased expression of Ang‐(1‐7) (TG) in the brain. Sprague‐Dawley (control group ‐ CTL) and TG rats were instrumented with telemetry probe to measure BP (DataScience). In other animals, baroreflex bradycardia (through injection of phenylephrine) and cardiac autonomic tone (by sequential and alternating blockade of β‐adrenergic and muscarinic receptors) were assessed using direct BP recordings (Biopac System). TG rats showed a lower basal mean BP (97 ± 2 mmHg vs 103 ± 2 mmHg in CTL, during the day and 101 ± 2 mmHg vs 106 ± 2 mmHg in CTL, during the night, n=13‐15), without a significant alteration in baseline HR. However, TG rats presented an increased cardiac sympathetic tone (156 ± 6 beats/ min vs 94 ± 10 beats/ min, in CTL, n=6) without change the intrinsic HR. There was a trend to a decrease in vagal tonus of TG rats (31 ± 7 beats/ min vs 53 ± 11 beats/ min in CTL; p<0.15, n=6). In addition, no significant changes in the baroreflex bradycardia was observed. These data suggest that TG rats with a life time increase in Ang‐(1‐7) in the brain present an important change in BP and a direct or compensatory change in autonomic activity.

Grant Funding Source: Support by PRONEX and INCT‐Nanobiofar Grants (CNPq and FAPEMIG) and CAPES
Renal responses to intra‐arterial infusion of a peroxynitrite scavenging agent with or without angiotensin II in anesthetized rats (1134.4)
Authors: Majid Dewan , Castillo Alexander
Published: June 2021

Peroxynitrite (OONO‐) is continually formed in the biological tissue by the spontaneous reaction between nitric oxide and superoxide. However, its potential role in the regulation of renal hemodynamics and excretory function in control condition as well as in the condition of elevated angiotensin II (AngII) level is not yet clearly defined. In the present study, we examined the responses to a OONO‐ scavenging agent, mercaptoethyl guanidine (MEG; 5 µg/kg/min for 75 min) infused directly into the left renal artery in anesthetized (Inactin; 100 mg/kg; i.p.) rats pretreated with (n=5) or without (n=5) AngII (1 ng/kg/min). The MEG dose was effective in blocking the increased renal blood flow (RBF) response to a bolus dose infusion of OONO‐ (40 µg/kg). RBF was recorded by the Transonic flow probe placed on the left renal artery and glomerular filtration rate (GFR) was determined by inulin clearance. In control rats (n=5), MEG infusion did not alter renal vascular resistance (RVR), RBF or GFR but caused significant decreases in urine flow (V, 9.5±1.4 to 7.8±1.5 μL/min/g; ‐20.2±6.7%), sodium excretion (UNaV, 1.69±0.38 to 1.34±0.43 μmol/min/g; ‐29.3±14.2%) and in fractional excretion of sodium (FENa, 1.04±0.33 to 0.87±0.36%; ‐29.3±15%). In a separate group (n=5), AngII treatment caused an increase of 18.0±4.1% in RVR (baseline value, b; 14.8±1.9 mmHg/mL/min/g) and the decreases of 14.4±3.6% in RBF (b, 7.2 mL/min/g), 17.1±6.2% in V (b, 6.7±2.3 μL/min/g), 35.6±14.4% in UNaV (b, 1.17±0.61 μmol/min/g) and 37.4±12.5% in FENa (b, 0.9±0.6%) without appreciable change in GFR. However, MEG infusion in these AngII treated rats caused similar decreases in renal excretory parameters (‐19.7±10.9% in V, ‐31.8±13.4% in UNaV and ‐33.3±8.4% in FENa) without significant changes in RVR, RBF or GFR. These data indicate that the endogenous OONO‐ exerts minimal hemodynamic effect but contributes to the maintenance of renal excretory function both in control condition as well as in the condition of elevated AngII level.
Differential blood pressure response in normotensive rats and rats with angiotensin II‐dependent hypertension following systemic cannabinoid CB1 receptor blockade (1140.11)
Authors: Schaich Chris , Howlett Allyn , Brosnihan K. , Diz Debra
Published: June 2021

Evidence for interactions between the endocannabinoid (ECS) and the renin‐angiotensin (RAS) systems in brain and vasculature is emerging. In transgenic (mRen2)27 rats, an angiotensin (Ang) II‐dependent model of hypertension involving excess sympathetic nervous system activity (SNS), acute systemic blockade of CB1 cannabinoid receptors by the CB1‐selective antagonist SR141716A (SR; 10 mg/kg orally; n = 5) significantly reduced systolic blood pressure (SBP) from 176 ± 3 mmHg baseline to 134 ± 3 mmHg after 90 min (P < 0.001). SBP showed evidence of recovery after 24 h but remained significantly reduced at 146 ± 5 mmHg (P < 0.01) compared to baseline. Plasma collected at 24 h showed no differences in levels of Ang II and Ang‐(1‐7) in the SR‐treated group compared to the vehicle (0.1% Tween‐80 in dH2O) group. Interestingly, oral SR treatment in normotensive Sprague‐Dawley control rats (n = 2) modestly increased SBP from a baseline of 120 ± 3 mmHg to 135 ± 2 mmHg after 90 min, suggesting blockade of the known vasodilator actions of the CB1 receptor. After 24 h, SBP recovered to 126 ± 4 mmHg. There was no effect of vehicle on SBP in either strain. These results indicate that CB1 receptor blockade in (mRen2)27 rats may disrupt the hypertension by interfering with the upregulated RAS‐dependent excess SNS activity in these animals, overcoming the blockade of the beneficial ECS effects in the vasculature.

Grant Funding Source: Supported by NIH: P01‐HL051952 and R01‐DA03690
Permethrin, a pyrethroid insecticide, impairs insulin‐stimulated glucose uptake in C2C12 myotubes (1142.7)
Authors: Kim Jonggun , Park Yooheon , Yoon Kyong , Clark John , Park Yeonhwa
Published: June 2021

Pyrethroids are a class of insecticides structurally derived from the naturally occurring insecticides called pyrethrins originating from Chrysanthemum flowers. Based on emerging evidence that exposures to pyrethroid insecticides were linked to altered glucose homeostasis, we investigated the role of permethrin (a pyrethroid insecticide) on glucose uptake using a C2C12 myotube model. C2C12 myoblasts were differentiated into myotubes with 0, 10, and 20 μM permethrin for 8 days. Permethrin treatment significantly reduced insulin stimulated glucose uptake in this model. Further, we found that permethrin affected insulin‐stimulated phosphorylation of Protein Kinase B (AKT) without altering total AKT levels, targeted at both Serine (Ser 473) and Threonine (Thr 308) sites. Permethrin inhibited insulin‐stimulated activation of phosphoinositide‐dependent kinase (pPDK), one upstream regulator of phosphorylation on AKT at Thr 308, without influencing activation of phosphatase and tensin homolog (pPTEN). These data suggest that permethrin impaired insulin responses by influencing AKT signaling by regulating two independent pathways, upstream of PDK/Thr 308 AKT and regulator(s) of Ser 473 AKT.
Does agomelatine have antidepressant action in juvenile rats? (1144.4)
Authors: Young Michael , Middlemas David
Published: June 2021

The purpose of this project is to investigate the effects of agomelatine on hippocampal neurogenesis and behavior in juvenile rats, which may lead to testing agomelatine for treating juvenile depression. Postnatal day 21 (PND21) juvenile Sprague‐Dawley rats were divided into 2 subgroups: 1) neurogenesis, and 2) behavioral. Each subgroup was further divided into experimental and control groups and injected intraperitoneally (IP) with agomelatine (10 mg kg¯¹) in 1% hydroxyethyl cellulose or vehicle respectively. The neurogenesis subgroup was injected once daily for 14 days. The behavioral subgroup was injected for 3 days, the first 2 days according to a porsolt forced swim test protocol, and once on the third day. The neurogenesis subgroup was also injected IP with BrdU three times a day for two days at the end, mitogenic, or the beginning, cell survival, of the 14 day injection period. After BrdU administration, the rats were sacrificed, and brain tissues fixed. The fixed brains were sectioned and analyzed using immunohistochemistry to detect BrdU and NeuN to assess differences in hippocampal mitogenesis and neurogenesis. The behavioral subgroup was subjected to a porsolt forced swim test to predict antidepressant activity, an open field test to detect locomotor differences, and an elevated plus maze to detect anxiety differences. Preliminary results show behavioral and structural trends that may be characteristic of an antidepressant effect. The results of the forced swim test showed that agomelatine decreased immobility time, which predicts agomelatine may be an antidepressant. There was not a significant difference between groups in the open field and elevated plus maze tests. The preliminary immunohistochemistry analysis of mitogenesis demonstrated an increased number of hippocampal cells in the subgranular zone (SGZ) and hilus. The analysis of neurogenesis with 10 mg kg¯¹ of agomelatine is underway. Agomelatine may act as an antidepressant drug in juvenile rats.
Alterations in brain‐derived neurotrophic factor following withdrawal from a chronic, escalating dose regimen of d‐amphetamine (1145.2)
Authors: Fuller Jasmine , Murray Ryan , Horner Kristen
Published: June 2021

Withdrawal from chronic, escalating doses of D‐amphetamine (AMPH) induces a syndrome with symptoms that are similar to those seen in major depressive disorder (MDD), such as dysphoria and anhedonia. These changes in affect may underlie relapse and the maintenance of addiction. Changes in brain‐derived neurotrophic factor (BDNF) may underlie the pathophysiology of MDD, but it is unclear whether alterations in BDNF occur following withdrawal from a chronic, escalating dose regimen of AMPH. The goal of this study was to examine changes in BDNF after exposure to chronic, escalating doses of D‐AMPH, followed by 24h or 4d of withdrawal. Animals were sacrificed, the tissue processed for immunohistochemistry and changes in BDNF examined in the nucleus accumbens, amygdala, hippocampus, dorsal raphe and prefrontal cortex, which are regions that mediate emotion and are impaired following psychostimulant withdrawal. Our data show that at 24h post‐AMPH BDNF protein levels are increased in the nucleus accumbens and decreased in the hippocampus. At 4d post‐AMPH, BDNF protein levels are significantly decreased in all areas examined. Our data suggest that while some of the initial dysphoric/anhedonic effects of AMPH withdrawal, mediated by increased BDNF in the nucleus accumbens, may abate as withdrawal progresses, other impairments, mediated by widespread decreases in BDNF may persist in longer‐term withdrawal from AMPH.

Grant Funding Source: Mercer Univeristy Seed Grant
In vitro pharmacology of pipradrol derivatives, 3,4‐methylenedioxypyrovalerone, and naphyrone (1145.3)
Authors: Liechti Matthias , Simmler Linda , Rickli Anna , Hoener Marius
Published: June 2021

Objective: The aim of the study was to characterize the in vitro pharmacology of novel psychoactive substances (NPS) found in “ivory wave” including the methylphenidate‐like pipradrol derivatives desoxypipradrol (2‐diphenylmethylpiperidine, 2‐DPMP) and diphenylprolinol (diphenyl‐2‐pyrrolidinemethanol, D2PM) as well as the pyrovalerone cathinones 3,4‐methylenedioxypyrovalerone (MDPV) and naphyrone (bath salts). Methods: We assessed 3H‐monoamine uptake and reverse transport in HEK 293 cells expressing the human serotonin‐, norepinephrine‐, or dopamine reuptake transporters (SERT, NET, or DAT, respectively). Binding affinities at monoaminergic receptors were assessed using displacement of radioactive ligands. Results: D2PM and 2‐DPMP were selective catecholamine transporter inhibitors without transporter‐mediated substrate‐releasing properties similar to methylphenidate. 2‐DPMP was an equally potent DAT/NET inhibitor to methylphenidate, while D2PM was less potent. Compared with classical stimulants, 2‐DPMP was a 10‐fold more potent DAT blocker compared with cocaine. The pharmacological profile of the pipradrol‐drivatives was similar to that of the pyrovalerone cathinones MDPV and naphyrone. MDPV very potently inhibited NET and DAT but not SERT. Naphyrone was equipotent at all transporters. Both MDPV and naphyrone did not release monoamines. None of the compounds exhibited relevant affinity to monoaminergic receptors. Conclusion: Taken together the pipradrol derivatives and the pyrovalerone cathinones are potent and with the exception of naphyrone selective catecholamine uptake inhibitors in line with their potent and prolonged psychostimulant actions. The pharmacological profile is also likely associated with high abuse liability and an increased risk of psychiatric complications.

Grant Funding Source: Swiss National Science Foundation