bluuebunny/crossref_metadata_2025 huggingface dataset:
Aortic vasorelaxation is not blunted in female Zucker diabetic fatty rats and is unaltered by anti‐diabetic metformin treatment, chronic exercise training, or a combination of both
Authors: Graham Drew A. , Smith Angela C. , Dyck David J. , Rush James W.E.
Published: June 2021

Male (m) Zucker diabetic fatty rats (ZDF) exhibit severely impaired endothelium‐dependent vasorelaxation (EDR) but responses in female (f) ZDF are unknown. fZDF (6 wk old, n=66) received 8 wk of: A. standard non‐diabetogenic chow (16.7 % kcal from fat) with no intervention and were lean (Lean, +/?, 188±3 g) or obese (Con, fa/fa, 358±6 g); or B. high‐fat diabetogenic chow (48 % kcal from fat, all fa/fa) with either no intervention (HF, 345±8 g), anti‐diabetic metformin treatment (HF‐Met, up to 500 mg/kg/d, 350±7 g), chronic exercise training (HF‐Ex, up to 2 h/d, 18–19 m/min, 10 % grade, 5 d/wk, 339±3 g), or Met and Ex (HF‐E+M, 337±4 g). In vitro thoracic aortic EDR (acetylcholine, 10−10−10−4 M) was assessed in the absence (ND) or presence of a superoxide dismutase mimetic (Tempol, 10−4 M). All fa/fa groups were obese and hyperinsulinemic vs. Lean (p<0.05). Hyperglycemia induced in HF was abrogated by all interventions (p<0.05). EDR was equal and unimpaired in all groups in the ND condition. Tempol elevated EDR in HF‐Ex (vs. ND, p<0.05), but did not affect any other groups. In this first report of vasomotor function in fZDF, responses were unaffected by severe obesity and diabetic‐like symptoms and unaltered by Met and/or Ex treatment. These data contrast the severely impaired EDR in mZDF. Mechanism(s) responsible for the apparent sex‐dependent vasculoprotection in fZDF is presently unclear. Funded by HSF Canada, NSERC, and CIHR.
Characterization of receptor‐ and store‐operated cation currents in rat pulmonary arterial smooth muscle cells (PASMCs)
Authors: Lin Mo‐Jun , Yang Xiao‐Ru , Sham James S.K.
Published: June 2021

Store‐ and receptor‐operated cation channels (SOC and ROC) in PASMCs have been implicated to play important roles in many physiological functions, including hypoxia‐ and agonist‐induced vasoconstriction, and pulmonary hypertension. Here we characterized the SOC and ROC currents in rat intralobar PASMCs. PASMCs were whole‐cell voltage‐clamped. K+ and Ca2+ currents were eliminated by substituting K+ with Cs+, and by nifedipine. Depletion of SR Ca2+ stores with cyclopiazonic acid activated a small inward current at −60 mV. The current was elicited with a time‐delay, exhibited typical bimodal dependence to extracellular Ca2+ due to the effect of anomalous mole fraction, and was blocked by La3+ or substitution of Na+ with N‐methyl‐D‐glucamine. I‐V relationship of La3+‐sensitive SOC current was quasi‐linear with a reverse potential close to 0 mM, indicative of its non‐selective nature. ROC agonist 1‐oleoyl‐2‐acetyl‐sn‐glycerol also activated a La3+ (1 mM) sensitive non‐selective cation current with slight outward rectification. Moreover, ROC currents recorded in PASMCs of chronic hypoxic rats (10% O2, 4 weeks) were significantly larger than those of normoxic rats. These results are consistent with the notion that functional SOC and ROC are present in PASMCs and their activities are up‐regulated in hypoxic pulmonary hypertension. (Supported by NIH R01HL074134, NSFC 30670772 and NSF(Fujian) C0620002)
N‐methyl‐D‐aspartate (NMDA) increases cortical blood flow in part by evoking cortical spreading depression (CSD) in insulin resistant Zucker rats
Authors: Lenti Laura , Domoki Ferenc , Bari Ferenc , Busija David W
Published: June 2021

Insulin resistance (IR) leads to selective impairment of responsiveness of larger cerebral arteries but effects on total cortical blood flow (CoBF) have not been examined. The purpose of our study was to test whether NMDA‐induced cerebral vasodilation is altered in the Zucker obese rat, a genetic model of IR. Anesthetized, ventilated, 12 week old male Zucker obese (n=8) and lean (n=8) rats were equipped with closed cranial windows. NMDA (10‐4, 10‐3 M) was applied to the parietal cortex, while CoBF (Laser‐Doppler flowmetry) and cortical DC potential were monitored. NMDA evoked CSD often at 10‐4 and always at 10‐3 M concentration. The amplitude of the response was similar in the two groups at all doses. After the transient CSD‐induced hyperemia, sustained increases in CoBF occurred with both 10‐4–10‐3 M NMDA at 6–10 minutes and there were no differences between obese and lean rats. Increases in CoBF during application of 10‐4 and 10‐3 M NMDA (% of baseline, 6–10th min of NMDA, *p<0.05 compared to vehicle) were 12±6 and 40±9*% in lean and 19±8% and 40±8*% in obese rats. Thus, similar to mice, the total cerebral vascular response to NMDA is due to direct effects by individual neurons as well as evoked CSDs. The retention of normal increases in CoBF in obese rats to NMDA/CSD indicates microvascular responses to some stimuli are intact in IR animals. Supported by the NIH (HL30260, HL65380, HL77731) and by the OTKA (K68976 and K63401).
Downregulation of mechanosensitive βENaC and γENaC in VSMCs in ASIC2 +/− mice
Authors: Gannon Kim P , VanLandingham Lauren G , Drummond Heather A
Published: June 2021

Previously, we evaluated the importance of the mechanosensitive protein ASIC2 in pressure‐induced constriction (PIC) in middle cerebral arteries in the ASIC2 mouse model. ASIC2 +/+ and −/− mice developed similar PIC (16 ± 2 and 10 ± 3%, respectively at 90 mm Hg). However, surprisingly, ASIC2 +/− mice developed less PIC (4 ± 2%). Since previous reports indicate 1) β and γENaC also mediate PIC and 2) ENaC can interact with ASIC2, the goal of our study was to determine if reductions in mechanosensitive γ and γENaC protein might contribute to loss of PIC in ASIC2 +/− mice. To address this, we used quantitative immunolabeling for ASIC2, βENaC and γENaC in VSMCs from ASIC2 +/+, +/− and −/− mice. Fluorescence intensity (FI) was normalized for α‐actin and background subtracted (Fig. 1).

We found β and γENaC were suppressed in +/−, and γENaC upregulated in −/− mice VSMCs, suggesting an interaction between βENaC, γENaC and ASIC2. Next, we used co‐immunoprecipitation and confirmed a biochemical interaction among βENaC, γENaC and ASIC2 in isolated vessels. These findings suggest the loss of PIC in ASIC2 +/− mice may also be dependent on inhibition of VSMC β and γENaC expression and recovery of PIC in ASIC2 −/− mice may be due to compensatory upregulation of γENaC. Our findings support the hypothesis that mechanosensitive ion channels initiating pressure‐induced constriction in VSMCs include βENaC, γENaC and ASIC2. Supported by AHA and NHLBI.
Occlusive, diffuse coronary artery disease in Ossabaw miniature swine with metabolic syndrome
Authors: Neeb Zachary Paul , Edwards Jason M , Bratz Ian N , Alloosh Mouhamad A , Schultz Carrie , Thompson Kristi , Cunha Timothy , Sturek Michael
Published: June 2021

Coronary artery disease (CAD) is increased in patients with metabolic syndrome (MetS), which is a cluster of risk factors. Ossabaw miniature swine replicate all features of MetS when fed excess kcal atherogenic diet high in trans fat/cholesterol/fructose. We hypothesized that Ossabaw swine fed mixed fatty acid atherogenic diet will exhibit more severe MetS and CAD. Lean pigs (N=9) were compared to pigs that developed MetS after 22 weeks of high cholesterol/fructose diet with trans fat (TMetS; N=8) or mixed fat (hydrogenated oil, coconut oil, lard) (MMetS; N=6). Triglycerides, LDL cholesterol, and LDL/HDL ratio in MMetS (85 ± 20; 284 ± 37 mg/dL; 5.2 ± 0.9, respectively) were profoundly increased vs. TMetS (39 ± 2; 130 ± 28 mg/dL; 2.0 ± 0.3, respectively) and lean (23 ± 4; 14 ± 3 mg/dL; 0.3 ± 0.1, respectively). MMetS and TMetS were hypertensive, but MMetS was more insulin resistant vs. TMetS and lean. Lean had virtually no CAD. TMetS CAD was mostly fatty streaks in proximal segments. Strikingly, MMetS CAD was occlusive (44% stenosis), diffuse (59% wall coverage in distal segments), and complex, including calcification. CONCLUSION: Ossabaw swine that develop MetS on mixed fat atherogenic diet have profound dyslipidemia and CAD. Support: RR013223, HL062552, Purina TestDiet.
Galectin 3‐binding protein and clinical outcomes in patients with angiographically significant coronary artery disease
Authors: Gleissner Christian Albert , Smalley David M. , Keeley Ellen Catherine , Ley Klaus
Published: June 2021

Galectin‐3 binding protein (G3BP) serum levels have been associated with cancer, viral infection and autoimmune disease. We found elevated G3BP levels in monocyte‐derived foam cells (RNA) and plasma‐ but not platelet‐derived microparticles (protein). We hypothesized that G3BP may represent a biomarker of severity and outcome in coronary artery disease (CAD).

G3BP serum levels were measured by ELISA in 96 patients undergoing coronary angiography (58 male, age 63.6 ± 11.2 years), 79 of which had significant CAD (≥70% stenosis of epicardial vessels). Clinical follow‐up (up to 5 months) included myocardial infarction (MI), the need for revascularization, congestive heart failure, and death. Statistical analyses comprised Kruskal‐Wallis test, Pearson correlation, and multivariate analyses.

The mean G3BP serum level was 10.37±6.5 mg/l. In multivariate testing, low G3BP levels predicted the combined outcome of the need for percutaneous or surgical revascularization in patients taking lipid‐lowering agents (OR 0.38, CI 0.17–0.87, p = 0.022), and this relationship remained strong after adjusting for CRP level (OR 0.46, CI 0.21–1.01, p = 0.052). Similar trends were seen for the combined endpoint of death or MI in patients with hypertension (OR 0.52, CI 0.25–1.10, p = 0.089), diabetes (OR 0.42, CI 0.17–1.06, p = 0.067), and those taking lipid‐lowering agents (OR 0.42, CI 0.15–1.16, p = 0.093).

In summary, we propose that G3BP is a promising biomarker that appears to have predictive value in addition to that of CRP in patients with significant CAD. Additional studies analyzing larger numbers of patients will be needed to assess it potential role as a biomarker.
DOES PERIVASCULAR FAT MODULATE CORONARY ARTERY CONTRACTILITY?
Authors: Paul Richard J. , Bowman Peggy Sue , Parr Anthony J.
Published: June 2021

Adipose tissue is reported to modulate contractility in vascular smooth muscle, via a postulated relaxing factor (ADRF) derived from adipocytes. We studied pig LAD coronary arteries with or without endothelium (+/−E) and/or fat (+/−F). +E preparations produced greater forces (P<0.05), whereas +F preparations reduced force for KCl, Histamine and U46619, but not for ACh. The effects of fat were concentration‐dependent for +E and greatest at submax [U46619]. A role for endothelin‐1 was tested with U46619, using an ET receptor antagonist, PD145065 (1 μM). The COX inhibition with indomethacin (1E‐5M) was used to test for potential ADRF's. There were no changes in force after PD145065. Relaxation to indomethacin was also similar in endothelial and de‐endothelialized LAD, suggesting eicosanoids are not involved. Indomethacin significantly relaxed all LAD, but there was marked decrease in relaxation by LAD with fat indicating a potential prostanoid nature for ADRF. Our data suggest the presence of both an endothelial constricting factor and an ADRF and interactions between adipose tissue and the endothelium. Supported by AHA 0655300B and NIH HL61974(RJP).
Fatty acid hydroperoxide increases ROS production and induces cytochrome C release in skeletal muscle mitochondria
Authors: Bhattacharya Arunabh , Liu Yuhong , Muller Florian , Perez Viviana , Jang Youngmok Charles , Ran Qitao , Van Remmen Holly
Published: June 2021

Inflammation and oxidative stress have been linked to several diseases including neurodegenerative and cardiovascular diseases. The purpose of this study was to determine the effect of the inflammatory mediators (15‐HPETE/15‐HETE; derived from the 15‐lipoxygenase (LOX) pathway) on muscle mitochondrial ROS production as measured by the Amplex Red assay. 15HPETE (and not 15HETE) increased ROS generation with complex I linked substrates, glutamate/malate, pyruvate/malate (nearly 2‐fold) but not with complex II linked substrate, succinate. Inhibiting reverse electron transfer with rotenone increased ROS with these substrates. 15HPETE inhibited complex I activity 10–25%, dose dependently but had no effect on complex II activity. In contrast, 15‐HETE had no effect on ROS or complex activities. In addition, 15‐HPETE induced ROS production was decreased 35–40% in mitochondria isolated from glutathione peroxidase 4 transgenic mice. 15HPETE induced increased release of cytochrome C and apoptosis inducing factor in isolated mitochondria suggesting increased susceptibility to apoptosis. We hypothesize that these effects may be reversed in GPX4Tg mice. In a denervation model of muscle atrophy, 15‐LOX inhibitor, PD146176 inhibited ROS production by ∼ 40%. Taken together, our results suggest a role of LOX pathway and fatty acid hydroperoxides in mitochondrial dysfunction and muscle atrophy.
Homocysteine modulates Gq11, G12/13, Gi/o G protein expression independent of protease activated receptor (PAR)
Authors: Vacek Thomas Paul , Tyagi Neetu , Sen Utpal , Moshal Karni , Passmore John , Kumar Munish , Steed Mesia , Tyagi Suresh
Published: June 2021

Homocysteine (Hcy) indirectly activates protease‐activated receptors (PARs). PARs belong to the G protein‐coupled receptor family (GPCR) that utilizes Gq/11, G12/13 and Gi/o. Differential expression of these G proteins is involved in cardiac hypertrophy. Since agonist binding to PARs induces desensitization and sequestration of the PAR receptors, we hypothesize that the intracellular G protein content will be modulated in relation to PAR receptor. Microvascular endothelial cells (MVECs) were treated with varying concentrations of Hcy 0–100μM for 0–48 h. RT‐PCR was used to determine transcription levels of PAR receptors. Expression of the four PAR receptors and their G proteins, Gq/11, G12/13, Gi/o, was determined with Western blot analysis. We report that PAR‐2 and PAR‐3 transcription decreased, while PAR‐4 transcription levels increased in a time and concentration‐dependent manner. G q/11 G protein content decreased at 24h. G12/13 and Gi/o expression was decreased at 48h with higher Hcy concentration. However, protein expression levels of all PAR receptors remained the same at 24 h and 48 h. Confocal analysis confirmed differential expression of Gq/11,G12/13 and Gi/o. The results suggest that differential G protein expression is not attributed to PAR receptor modulation. This suggests that Hcy modulates the down‐regulation of other GPCRs that utilize these G proteins.
Function of V‐ATPase as a putative endosomal pH‐senor: mapping the interaction sites between V‐ATPase a2‐isoform and ARNO.
Authors: Merkulova Maria , Marshansky Vladimir
Published: June 2021

Previously we demonstrated that V‐ATPase a2‐isoform directly interacts with ARNO. The a2‐isoform is specifically targeted to early endosomes and a2/ARNO interaction is modulated by acidification of the endosomal lumen. This acidification‐dependent process is crucial for trafficking within the endosomal/lysosomal protein degradative pathway. We propose that the V‐ATPase a2‐isoform functions as an endosomal pH‐sensor. However, the molecular mechanism of this novel function of V‐ATPase and its interaction with small GTPases remain obscure. Here we mapped the interaction sites between a2‐isoform and ARNO. First, using recombinant constructs containing various ARNO domains we demonstrated that cytosolic N‐terminal tail of the a2‐isoform (a2N) specifically interacts with plekstrin‐homology domain of ARNO. Next, we synthesized 22 peptides which are covering the complete sequence of a2N. Peptide pull‐down assay demonstrated that ARNO strongly interacts with one (a2N‐03) and weekly interacts with three (a2N‐11, a2N‐12 and a2N‐18) peptides. Interaction of a2N‐03 with ARNO was specific since it does not occur with GST. Based on these data, the first model of the folding of V‐ATPase a2‐isoform is proposed. We also suggest the two‐step mechanism of interaction of a2‐isoform with ARNO during which some conformational changes are taking place. This study was supported by NIH DK038452 and BADERC DK057521 grants.
Interaction between STIM1 and TRPC1/TRPC4 complexes, a mechanism for store‐operated calcium entry in glomerular mesangial cells
Authors: Sours‐Brothers Sherry , Ding Min , Graham Sarabeth , Ma Rong
Published: June 2021

Store‐operated Ca2+ entry (SOCE) participates in a diverse array of cell functions in a variety of cell types. Although recent evidence suggests that Orai proteins might be the molecular entity of store‐operated channels (SOC) in hematopoietic cells, multimers of canonical transient receptor potential (TRPC) proteins are still candidates of SOC in other types of cells. Our previous studies demonstrated that TRPC1 and TRPC4 are expressed and interact with each other in glomerular mesangial cells (MCs). The present study examined if complexes of TRPC1/TRPC4 proteins mediated SOCE in MCs and if activation of the channel complexes required STIM1, a Ca2+ sensor protein in the endoplasmic reticulum. Knockdown (KD) of TRPC1 or TRPC4 expression using RNAi inhibited an increase of channel open probability stimulated by store‐depletion with thapsigargin (TG), as measured by cell‐attached patch clamp. TRPC1 and TRPC4 interaction, as detected by co‐immunoprecipitation (co‐IP), was enhanced by TG treatment. KD of TRPC1 or TRPC4 also attenuated SOCE, measured by fluorescent Ca2+ imaging, while simultaneous KD of both subunits had a significantly greater effect. Furthermore, association of STIM1 with TRPC4 was also found by co‐IP, and KD of STIM1 inhibited TG‐induced SOCE. These data imply that TRPC1 and TRPC4 form a complex that is activated by store‐depletion. STIM1 also interacts with this complex and is required for SOCE.
Phosphoproteomics of Human Urinary Exosomes
Authors: Gonzales Patricia A , Pisitkun Trairak , Hoffert Jason , Star Robert A , Wang Nam Sun , Knepper Mark A
Published: June 2021

Urinary exosomes are the low‐density membrane vesicles that originate from the internal vesicles of multivesicular bodies and are secreted into urine by every renal tubule cell types as well as glomerular podocytes. Urinary exosomes are known to contain an extensive repertoire of proteins including many potential biomarkers. These vesicles are isolated and purified by differential centrifugation from urine. In this study, we sought to define the phosphoproteome of human urinary exosomes using a combination of phosphopeptide enrichment by immobilized metal affinity chromatography and phosphorylation site identification by LC‐MSn neutral loss scanning. Nineteen phosphorylation sites corresponding to fourteen unique phosphoproteins were identified in exosomal proteins from human urine samples. The identifications include phosphorylation sites previously identified in AQP2 (S256), NKCC2 (T118 and S120), CHMP2B (S199), HSP90AB1 (S255) and SPP1 (S192, S197, and S207). Novel phosphorylation sites were identified on the following proteins: GPRC5B, GPRC5C, RRAS2, VPS4B, cytochrome b reductase, proteasome alpha 3, mucin 1, and the sodium chloride cotransporter (NCC). NCC was phosphorylated at S811. We conclude that human urinary exosomes contain many phosphoproteins. This study demonstrates the potential for identification of novel phosphoproteins from human urinary exosomes using shotgun phosphoproteomic analysis.
Fetal programming increases susceptibility to renal injury induced by ischemia‐reperfusion in IUGR offspring
Authors: Ojeda Norma Beatriz
Published: June 2021

Renal injury due to ischemia‐reperfusion (I‐R) is the major cause of acute renal failure. Whether susceptibility to renal injury from I‐R can be programmed during fetal life is unknown. Our laboratory has developed a unique rat model of placental insufficiency that results in intrauterine growth restriction (IUGR) and development of hypertension in IUGR rats. We hypothesized that IUGR will also lead to greater susceptibility to renal injury following I‐R. Physiological parameters were determined 1 week after mild renal ischemia (15 min); early histological renal changes were assessed in a subset of rats after 30 min of reperfusion. Mean arterial pressure (MAP), renal vascular resistance (RVR), and histological markers of renal damage were increased; glomerular filtration rate (GFR) and renal blood flow (RBF) were decreased following I‐R in IUGR compared to control (Table). Therefore, these findings suggest that influences during fetal life can program an increased susceptibility to renal injury suggesting IUGR individuals may be an ‘at risk’ population for renal disease.
Contribution of renal epithelial sodium channel in sodium retention during chronic heart failure
Authors: Zheng Hong , Liu Xuefei , Rao U.S. , Patel Kaushik P
Published: June 2021

The goal of the present study was to examine the contribution of renal epithelial sodium channel (ENaC) in the increased sodium and water retention during chronic heart failure (CHF). We hypothesized that over expression of ENaC could be involved in sodium retention during CHF. The left coronary ligation‐induced heart failure model in the rat was used. There was significant increase in diuretic (7‐fold of sham) and natriuretic responses (3‐fold of sham) to the ENaC inhibitor benzamil in the rats with CHF compared to sham rats. Real‐time PCR and Western blotting demonstrated that the mRNA and protein abundance of α, β, and γ‐subunits of ENaC were significantly increased in the cortex (α‐ENaC: mRNA Δ104±24%, protein Δ32±7% compared to sham rats) and outer medulla (α‐ENaC: mRNA Δ52±18%, protein Δ33±9% compared to sham rats) in CHF compared to sham rats. Immunohistochemical imaging confirmed the increased labeling of α‐ENaC, β‐ENaC and γ‐ENaC subunits in the collecting duct segments in rats with CHF compared to sham rats. These results suggest that the increased expression of renal ENaC subunits may contribute to the renal sodium and water retention observed during CHF. (Supported by NIH)
Sexual dimorphism in hypovolemic thirst induced by Furosemide and low sodium diet treatment (F/LSD)
Authors: Vivas Laura , Macchione Ana Fabiola , Dalmasso Carolina
Published: June 2021

In a two‐bottle free choice test we investigated the F/LSD‐induced water and 2% NaCl intake in female (F), ovariectomized (FOVX) and male rats (M). We also studied estrogen receptor α immunoreactivity (ER‐α ir)and Fos‐ir in the subfornical organ (SFO) and median preoptic nucleus (MnPO), key areas involved in body fluid homeostasis.

Before F/DBS treatment we found that salt preference (NaCl intake/NaCl + water intake) in F and FOVX was higher compared to M (p≤0.001). F/LSD‐induced depletion increased water intake in F versus FOVX and M (p≤0.001). However induced‐sodium appetite was similar in all groups.

In the SFO, F/LSD increased Fos expression in F and M, but decreased Fos‐ir neurons in FOVX compared to non‐depleted rats (p≤0.001).

Within the MnPO, Fos‐ir of control animals was higher in M and FOVX compared to F (p≤0.001). After F/LSD, Fos‐ir did not change in F, however we observed a decreased activation in both M and FOVX (p≤0.01).

Regarding ER‐α, F/LSD increased the expression along SFO and MnPO only in F (p≤0.001). Besides, we found no differences after the treatment in both M and FOVX.

Our data support previous evidence suggesting the MnPO as an inhibitor of water intake in males. We also shown the participation of ER‐αalong the SFO and MnPO after F/LSD treatment in females. In conclusion, we may suggest that hypovolemic thirst induced by F/LSD is a sexual dimorphic phenomenon.

Supported by ANPCyT and CONICET.
Hepatic Stearoyl‐CoA Desaturase‐1 expression is required for Carbohydrate‐Induced Adiposity and Hepatic Steatosis
Authors: Ntambi James Mukasa , Flowers Matthew T. , Sampath Harini , Chu Kiki , Liu Xueqing , Miyazaki Makoto
Published: June 2021

Stearoyl‐CoA desaturase is a delta‐9 desaturase that catalyzes the desaturation of saturated fatty acids into monounsaturated fatty acids, which serve as the preferred substrates for synthesis of numerous tissue lipids. It is also a critical regulator of energy metabolism and its expression is implicated in numerous human diseases that include obesity, non‐insulin‐dependent diabetes, hypertension, cardiovascular disease and immune disorders. We investigated the role of SCD1 in liver metabolism by generating mice with a liver‐specific knockout of Scd1 (LKO) using Cre‐lox technology. We found that unlike global SCD1 KO (GKO) mice, LKO mice fed high‐fat diet were not protected against obesity, hepatic steatosis, and whole‐body insulin resistance. On the other hand, LKO mice were protected from high‐carbohydrate diet‐induced adiposity and hepatic steatosis similar to GKO mice. The LKO mice displayed a marked decrease in the rate of lipogenesis with decreased expression of the nuclear contents of SREBP‐1 and ChREBP and their target genes. Oleate but not stearate supplementation to the high carbohydrate diet normalized adiposity, triglyceride secretion and hepatic lipogenesis of LKO mice. These results indicate that hepatic SCD1 expression, and thus oleate is required for carbohydrate‐induced adiposity and lipogenesis but loss of SCD1 activity in extra hepatic tissues is required to protect mice from high‐fat diet‐induced obesity and insulin resistance.

Supported by NIH grant DK 62388.
VEGF Receptor Inhibitor Directly Suppresses Proliferation and Migration of Breast Cancer Cells
Authors: Young Emily , Tan Wei , Covington Jordan , Johnson James Wes , Gu Jian‐Wei
Published: June 2021

Recent studies show that certain tyrosine kinase inhibitors target both tumor vasculature and tumor cell. We determine whether the mouse breast cancer cells (E0771) express VEGF receptor‐1 and 2, and whether VEGF receptor inhibitor (SU5416) directly inhibits proliferation and migration of E0771 Cells. Human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HASMC) were used as the positive and negative controls. RT‐PCR and Western blot showed that E0771 cells express VEGF receptor‐1 & ‐2 mRNA and protein. 3H‐thymidine incorporation was determined in those cells following exposure to VEGF (10 ng/ml), VEGF plus SU5416 (10 μmol/L) for 18 hrs. VEGF caused a 42% increase in proliferation of E0771 cells, compared to the control (n=6; P<0.01). There was a significant decrease in proliferation of E0771 cells treated with VEGF plus SU5416 vs. the control (65%, P<0.01). VEGF caused a 2‐fold increase in proliferation of HUVEC vs. the control (P<0.01), but its action was completely abolished by SU5416. Neither VEGF nor SU5416 exerted any effect on proliferation of cultured HASMC. Matrigel assay showed that SU5416 (10 μmol/L) inhibited 47% migration of E0771 cells vs. the control (P<0.01). These findings suggest that VEGF receptor inhibitor SU5416 can directly target the breast cancer cells that express VEGF receptor for inhibiting the proliferation and migration. (NIH/AA013821 & NIH/HL51971)
Parasympathetic Dysfunction and Cardiac Arrhythmias in Diabetic Mice
Authors: Park Ho‐Jin , Du Chuang , Georgescu Serban P , Madias Christopher , Zhang Yali , Aronovitz Mark J , Dunlap Kathleen , Galper Jonas B
Published: June 2021

Aim: to establish a molecular mechanism for diabetic autonomic neuropathy and the increased incidence of arrhythmia and sudden death in a mouse model for type I diabetes. In the heart acetylcholine binding to M2 receptors releases Gαi2 and inhibits β‐adrenergic stimulated cAMP levels and L‐type Ca currents (ICa, L). Isoproterenol (Iso) stimulated ICa, L equally in both ventricular myocytes from WT and type I diabetic Akita mice, 2.8±0.13 (N=13) and 2.71±0.27 (N=10) fold, respectively. However, carbamylcholine (Cab) inhibition of I so‐stimulated ICa, L was markedly reduced in Akita myocytes compared to that in WT, 30.4±2.8% vs 55.2±3.6% (p<0.01). Furthermore, Gαi2 expression in Akita ventricles was decreased to 39±12% of WT (p=0.02). In insulin treated euglycemic Akita mice, Iso stimulated ICa, L 3.23±0.3 (N=11) fold while carb inhibited ICa, L by 64.3±3.3% consistent with insulin reversal of parasympathetic dysfunction. Insulin stimulated Gαi2 expression in cardiomyocytes. Following myocardial infarction Akita mice developed 8,000 PVCs/hour and 4000 runs of ventricular tachycardia (VT) (N=15), peak 18h, compared to 50 PVCs/hour and no VT in WT mice (N=15). These data supported the conclusion that hypoinsulinemia in the type I diabetic mouse results in decreased expression of Gαi2, which leads to a relatively unopposed sympathetic input to the heart and predisposes the heart to life‐threatening arrhythmias.
Transduction of recombinant heat shock protein 27 increases contractility of vascular smooth muscle
Authors: Smoke Christopher C. , Morley Erica L. , Flynn Charles R. , Komalavilas Padmini , Brophy Colleen M.
Published: June 2021

Heat shock protein 27 (HSP27) is a stress induced cytoprotective protein, which has been shown be involved in contraction, stress fiber formation, and reorganization of actin cytoskeleton. In this study, the role of HSP27 on contractility in rat aortic vascular smooth muscle (VSM) tissue was examined by using a transducible recombinant HSP27 (rPTDHSP27). Human HSP27 cDNA, that was fused with a protein transduction domain and 6x Histidine tag at the N terminal end, was expressed in Escherichia coli and purified using a metal chelating affinity column. The purified protein was verified through Western blot and HPLC ESI MS/MS. VSM rings were equilibrated in a muscle bath, and contracted with norepinephrine (NE) (1–1000 nM) with or without 3.8 μM rPTDHSP27 and force generated was measured. Norepinephrine induced a dose dependent contraction, which was enhanced with the pretreatment of rPTDHSP27 (84.1% compared to 16.9% in the control with 16 nM NE). The concentration needed to produce maximal contraction was 10 fold lower when the tissue was pretreated with rPTDHSP27 (88% at 666 nM compared to 96% at 66 nM, for control and rPTDHSP27 treated rings respectively). NIH RO1 70715
Effect of calcitonin gene‐related peptide (CGRP) on avian appetite‐related processes
Authors: Calchary Wendy Alyssa , Cline Mark A
Published: June 2021

Calcitonin gene‐related peptide (CGRP), consisting of 37 residues, is found in the gastrointestinal tract and causes reduced feed intake in mammals. Its effects in the avian class are unreported. We found that intraceberoventricular (ICV) injection of CGRP caused a linear decrease in feed and water intake in Cobb‐500 chicks. We determined that the effect on water intake was secondary to feed since feed‐restricted chicks did not have reduced water intake. Next, we found that an intaperitonal (IP) injection of CGRP reduced feed intake, but did not affect water intake. In order to determine if the hypothalamus was associated with these affects we examined c‐Fos immnoreactivity in several key appetite‐related nuclei. Both ICV and IP CGRP caused activation of the nucleus dorsomedialis hypothalami and the nucleus ifundibuli hypothalamis, the homolog of the mammalian arcuate nucleus. The results demonstrated that CGRP causes anorexigenic effect in chicks and the hypothalamus is involved.
Changes in Hypothalamic a‐MSH in Rats Fed a High Whole Milk Protein Diet
Authors: Heppner Kristy Marie , Doane Dolores Fernandez , Guillon Marlene , Beverly J. Lee
Published: June 2021

The melanocortin system has been suggested to contribute to an increase in satiety associated with consumption of a high protein diet. To test this association, male Wistar rats were fed isocaloric diets containing either 14% (n=13) or 55% (n=8) whole milk protein (WMP) for two weeks. On day 14, food was removed during the light phase and returned at dark onset. Rats in lower protein group were provided with either the same diet (P14/P14, n=7) or the higher protein diet (P14/P55, n=6). All rats on the higher protein diet continued to receive that diet (P55/P55). Food intake was measured after 2 hr, at which time rats were euthanized and brains rapidly removed and frozen. Hypothalami were dissected and homogenized before measuring a‐MSH and orexin by RIA. During the 14 day prefeeding period, food intake for P55 was 18% lower than P14 (p<0.01); however, the amount of food consumed the 2 hr prior to euthanasia was similar (p=0.18) for all groups. There were no differences among groups in orexin (p=0.57). a–MSH concentrations (pg/mg protein) were also similar (p=0.16) among groups, though tended to be lower in rats fed the higher protein diet (P14/P14 = 36.5 ± 2.7 vs. P55/P55 = 28.7 ± 3.4) and intermediate in the P14/P55 group (33.1 ± 1.2). These results do not support an increase in a‐MSH activity contributing to greater satiety of a high protein diet when the protein source is WMP. Supported by the College of ACES James Scholar Honors Program.
Central α‐melanocyte stimulating hormone causes more potent anorexigenic effects in a line of chickens containing anorexic individuals as compared to a line that is obese
Authors: Bowden Christie Nichole , Nandar Wint , Siegel Paul B , Cline Mark A
Published: June 2021

α‐melanocyte stimulating hormone (α‐MSH) causes potent anorexigenic effects in both mammals and birds when centrally administered. We evaluated the central effects of α‐MSH in lines of chickens selected for low (LWS) or high (HWS) body weight for 49 consecutive generations. LWS contains some anorexic individuals while the HWS chickens are hyperphagic. Central α‐MSH more potently reduced feed intake in LWS than HWS. In a second experiment, we found that central α‐MSH reduced exploratory pecking in LWS but not HWS chicks. α‐MSH more potently reduced locomotion and increased deep rest in the LWS than HWS line. Although feed intake was reduced in LWS, their feeding efficiency was increased by central α‐MSH. In conclusion, LWS and HWS lines of chickens responded differently to central α‐MSH; the central melanocortin system may contribute to their differential body weights.
Angiotensin II inhibits GABAergic synaptic transmission in dorsolateral periaqueductal gray
Authors: Lu Jian , Xing Jihong , Li Jianhua
Published: June 2021

The purpose of this study was to determine the role of angiotensin II (Ang II) in modulating inhibitory and excitatory synaptic inputs to the dorsolateral periaqueductal gray (dl‐PAG). Whole cell voltage‐clamp recording was performed to obtain inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs) of the dl‐PAG neurons. Application of Ang II, in the concentration of 2 μM, decreased the amplitude of evoked IPSCs from 148±15 to 89±7 pA in eight tested neurons (P<0.05). The paired‐pulse ratio (PPR) of evoked IPSCs was also examined, and increased by 96±5% in control and by 125±7% after Ang II (P<0.05 vs. control). Additionally, Ang II decreased the frequency of miniature IPSCs from 0.83±0.02 to 0.45±0.03 Hz in ten tested neurons (P<0.05) without affecting the amplitude and the decay time constant. The effect of Ang II on miniature IPSCs was blocked by Losartan (an Ang II AT1 receptor antagonist), but not by PD123319 (an AT2 receptor antagonist). In contrast, Ang II wasn't observed to have distinct effects on the EPSCs. The data presented in this study provide strong evidence indicating that Ang II inhibits GABAergic synaptic inputs to the dl‐PAG through activation of presynaptic AT1 receptors (Supported by R01 HL075533, R01 HL078866 & R01 HL060800).
Genistein Inhibits Sympathetic Vasomotor Tone through Nitric Oxide and Tyrosine Phosphorylation in Rostral Ventrolateral Medulla
Authors: Wu Yu‐Min , Ma Hui‐Juan , Wang Xin , He Rui‐Rong , Li De‐Pei
Published: June 2021

Injection of 17β‐estradiol into the rostral ventrolateral medulla (RVLM) decreases sympathetic vasomotor tone. Phytoestrogen genistein inhibits protein tyrosine kinase and stimulates nitric oxide (NO) production. In this study, we determined the signaling transduction mechanisms underlying the sympathoinhibitory effect of genistein in the RVLM. Renal sympathetic nerve activity (RSNA), arterial blood pressure (ABP), and heart rate (HR) were recorded in urethane‐anesthetized rats. Bilateral microinjection of genistein (2, 20, and 200 pmol, 2 μl) into the RVLM dose‐dependently decreased RSNA, ABP, and HR. Pre‐microinjection of 200 pmol tamoxifen (a selective estrogen receptor antagonist) into the RVLM potentiated the inhibitory effects of genistein (20 pmol) on RSNA, BP, and HR. On the other hand, pre‐ microinjection of either 2 nmol sodium orthovanadate (an inhibitor of protein tyrosine phosphatase) or 200 pmol L‐NAME (a non‐selective nitric oxide synthesis inhibitor) into the RVLM attenuated the inhibitory effects of genistein on RSNA, BP, and HR. Furthermore, pre‐microinjection of a mixture of L‐NAME and sodium orthovanadate abolished genistein‐induced decreases in RSNA, BP, and HR. Collectively, these data provide these data provide novel information that nitric oxide and tyrosine phosphorylation are involved in the genistein‐induced sympathoinhibitory response in the RVLM.
Hyperpolarization‐activated cyclic nucleotide‐gated channels mediate blunted excitability of aortic baroreceptor neurons in diabetic rats
Authors: Li Yu‐Long , Tran T. Paul , Muelleman Robert , Schultz Harold D.
Published: June 2021

Although dysfunction of arterial baroreflex occurs in diabetic humans and animal models of type‐1 diabetes (T1D), the mechanisms involved in the impairment of the baroreflex still remain unclear. The nodose ganglia (NG) contain the cell bodies of the aortic baroreceptor (AB) neurons. Hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels are expressed in AB neurons and play an important role in regulating the excitability of these sensory neurons. We focused on the AB neurons in the NG to investigate whether the excitability of AB neurons is depressed and whether HCN channels are involved in this depression in streptozotocin (STZ)‐induced T1D rats, using the whole‐cell patch clamp technique. AB neuron excitability (action potential frequency) in the T1D rats was lower than that in the control rats (0.4 ± 0.6 vs 4.6 ± 0.7 spikes/sec, p<0.05; AB neurons were identified by DiI staining). In addition, HCN current density in AB neurons from the T1D rats was bigger than that from the control rats. Furthermore, HCN channel blockers (5 mM cesium chloride and 100 μM ZD‐7288) significantly reduced HCN currents and increased action potential frequency of the AB neurons in control and T1D rats. Using immunofluorescent and western blot analyses, we also found that the expression of HCN1 channel protein in the NG from the T1D rats was higher than that from the control rats. These results indicate that the HCN channels influence the excitability of AB neurons, and more importantly, contribute to the decreased excitability of AB neurons in T1D rats.
Unmyelinated anterior ethmoidal nerve fibers provide the afferent signal that induces activation of MDH neurons during nasal stimulation
Authors: McCulloch Paul , Hollandsworth Michael , DiNovo Karyn
Published: June 2021

The anterior ethmoidal nerve (AEN) innervates the nasal passages and constitutes the afferent limb of the cardiorespiratory reflex resulting from nasal stimulation. However it is currently unknown whether the AEN afferent signal is transmitted to the brainstem via unmyelinated or myelinated fibers. Our objective was to determine which of these fiber types connect with brainstem neurons activated during nasal stimulation. Initially AEN central projections were traced in the rat using WGA‐HRP (a non‐specific tracer), IB4‐HRP (specific for unmyelinated fibers) and CTB‐HRP (specific for myelinated fibers). Using TMB histochemistry, AEN terminal label was located ipsilaterally, ventrally and superficially within the medullary dorsal horn (MDH); in juxtaposition with the paratrigeminal nucleus (Pa5); and along the dorsal edge of the mesencephalic trigeminal nucleus (Me5). Unmyelinated fibers primarily project to MDH, while myelinated fibers primarily project to Pa5 and Me5. In other animals, after AEN tracer injections, nasal stimulation with ammonia vapors induced Fos production within activated neurons. Using fluorescent double‐labeling, unmyelinated fiber terminations clustered around Fos positive neurons within the MDH. We conclude that during nasal stimulation unmyelinated AEN fibers provide the afferent signal that induces activation of secondary trigeminal neurons within the MDH. Supported by NIH R15 HL080007.
Amygdala projecting solitary tract nucleus (NTS) neurons receive both direct and indirect cranial visceral afferent connections
Authors: McDougall Stuart J , Andresen Michael C
Published: June 2021

The solitary tract nucleus (NTS) is the central site of cranial visceral afferent termination within the CNS. Information arrives in the NTS via the solitary tract (ST) and is broadcast to other brain regions including the amygdala. Little is known about the organization and processing of such information by forebrain projecting NTS neurons that likely contribute to complex behaviors. We aimed to determine how cranial visceral afferent information reaches amygdala projecting NTS neurons. In horizontal slices (250 μm) from rats previously stereotaxically injected with rhodamine beads into the central nucleus of the amygdala, retrogradely labeled cell bodies were found throughout caudal NTS. Shocks to the ST evoked postsynaptic currents (PSCs) in labeled neurons. Some amygdala projecting NTS neurons exhibited constant latency ST‐evoked excitatory PSCs with no failures, indicating monosynaptic ST contacts. The remaining neurons exhibited multiple glutamatergic and GABAergic PSCs with highly variable latencies and frequent failures – findings consistent with polysynaptic ST pathways. These preliminary data suggest that cranial visceral afferent nerves, including the vagus, connect differentially to amygdala projecting NTS neurons.

Supported by: NHMRC of Australia (SJM ‐ C. J. Martin Fellow) and HL‐041119 (MCA).
Aerosolized capsaicin (CAP) pretreatment prevents lipopolysaccharide (LPS)‐induced inhibition of ventilatory responses to hypoxia and hypercapnia in rats
Authors: Zhuang Jianguo , Zhang Cancan , Xu Fadi
Published: June 2021

LPS causes pulmonary inflammation and blunted hypoxic (HVR) and hypercapnic ventilatory responses (HCVR) in anesthetized rats. Because CAP is a potent stimulant of bronchopulmonary C‐fibers (PCFs) and it promotes pulmonary inflammation, we hypothesized that the LPS‐induced inhibition of HCVR and HVR was aggravated by CAP pretreatment. Rats were exposed to either aerosolized CAP (0.4 mg/ml) or vehicle for 5 days for two 5‐min episodes daily that were 4 hr apart. After the pretreatment, the rats were intratracheally instilled with LPS (0.5 mg/0.5 ml) or saline, and anesthetized and tracheotomized 2 days later. Animals’ ventilatory responses to 10% O2 and 10% CO2 for 1 min and apneic response to right atrial bolus‐injections of CAP (1 and 4 μg/0.1 ml) were measured, and the bronchoalveolar lavage fluid (BALF) collected at the end of the experiments. We found that: 1) CAP and/or LPS pretreatment did not affect baseline ventilation; 2) LPS rather than CAP pretreatment alone inhibited HCVR (↓ 63%, P < 0.01) and HVR (↓47%, P < 0.05); 3) these LPS‐induced inhibitions disappeared in CAP‐pretreated rats; and 4) BALF total cell count was significantly increased by LPS, and this increase was not affected by coupling it with CAP pretreatment. We conclude that CAP pretreatment eliminates the LPS‐induced inhibitory effects on HVR and HCVR with little impact on LPS‐induced pulmonary inflammation. (Supported by NIH 74183).
The effects of sildenafil and acetazolamide on breathing efficiency during hypoxic exercise
Authors: Lalande Sophie , Snyder Eric M. , Olson Thomas P. , Hulsebus Minelle L. , Orban Marek , Somers Virend K. , Johnson Bruce D. , Frantz Robert P.
Published: June 2021

The ability to work efficiently at altitude is impaired by the reduced arterial oxygen tension. Acetazolamide may improve systemic saturation (SaO2) by stimulating ventilation but increases the work of breathing. Efforts have focused on the potential role of sildenafil in improving exercise performance in high altitude environment. The purpose of this study was to determine the effects of acetazolamide and sildenafil on oxygen uptake (VO2) and breathing efficiency during submaximal hypoxic exercise. Sixteen volunteers received sildenafil 40 mg (S), acetazolamide 125 mg (A) or matching placebo (P) tid × four doses in double‐blind fashion starting 8 hours prior to hypoxic exposure over 3 separate overnight stays in an altitude tent with an O2 concentration of 12.5%, and then performed hypoxic submaximal exercise on a stationary bicycle for 10 min at a workload of 40% of VO2peak. SaO2 decreased with exercise but there was no difference in exercising SaO2 or VO2 between conditions. As expected, acetazolamide increased ventilation compared to placebo and sildenafil. Breathing efficiency as measured by Ve/VCO2 decreased with acetazolamide in comparison to placebo and sildenafil (P: 42.6±1.2, S: 41.9±1.6, A: 46.7±1.1, p<0.01). Sildenafil did not change breathing efficiency while acetazolamide reduced breathing efficiency during hypoxic exercise. This study was funded by Mayo Foundation and NIH Grant No. HL71478.
PlGF is greater and sFLT‐1 lower in multigenerational vs. shorter‐term pregnant residents of high altitude
Authors: Davila Rita Daniela , Julian Colleen Glyde , Browne Vaughn A , Wilson Megan J , Hageman Jennifer Lynn , Yamashiro Henry , Rodriguez Armando , Rodriguez Carmelo , Vargas Enrique , Moore Lorna Grindlay
Published: June 2021

INTRODUCTION. An imbalance between angiogenic and anti‐angiogenic factors likely plays an etiological role in fetal growth restriction. Since multigenerational high‐altitude (HA) Andean (AND) vs. shorter‐term, European (EUR) ancestry protects against fetal growth restriction at HA, we hypothesized that the angiogenic factor PlGF was elevated and the anti‐angiogenic factor sFlt‐1 reduced. MATERIALS AND METHODS. Pregnant women residing at low (400 m; AND n=36, EUR n=39) or high (3600m; AND n=46, EUR n=33) altitude in Bolivia were studied at 20 and 36 wk of pregnancy and 4 mo postpartum. Plasma sFlt1 and PlGF levels were determined by ELISA and data analyzed by ANOVA. RESULTS. Pregnancy increased PlGF and sFlt‐1 in EUR and AND women (p<0.001). PlGF increased in AND women at HA in early pregnancy and declined at 36 wk compared EUR values (interaction between pregnancy, altitude and ancestry, p<0.05). At HA, sFlt‐1 was greater in EUR than AND women at 20 and 36 wk (p<0.001). The sFlt‐1/PlGF ratio rose from 20 wk to 36 wk in EUR women at both altitudes and in HA AND women but not in the LA AND, and tended to be lower in AND vs. EUR women near term (p<0.10). CONCLUSIONS. Lower sFLT‐1 and higher PlGF levels may help protect multigenerational AND compared with shorter‐term EUR HA‐residents from fetal growth restriction. (NIH HL079647 and HL 14985)
Altered thermal and metabolic control in newborn rats at high altitude
Authors: Joseph Vincent , Soliz Jorge , Gassmann Max , Isidro Marcelino Gonzales , Vargas Enrique , Soria Rudy
Published: June 2021

In acute hypoxia, newborn reduce oxygen consumption (VO2) and rectal temperature (Tr), but it remains unclear if these responses are maintained in chronic hypoxia. We used rats living at high altitude (3600 m, La Paz Bolivia) at postnatal days 7–8 (P7, n=5, body weight 13±1 g) and 15–16 (P15, n=6, 21±1 g). Tr and VO2 were measured in 21%O2 (room air), 35%O2 (sea level PO2), and 10%O2 (hypoxia), during 20 minutes each. Ambient temperature (Ta) was 34°C at P7 and 30°C at P15. Tr‐Ta was used as an index of thermoregulatory control. At P7, Tr‐Ta was −0.1±0.2°C, suggesting that thermoregulatory control is not established. In 35%O2, Tr was 34.8±0.2°C, and 32.0±0.6°C in 10%O2. At P15, Tr‐Ta was 5.2±0.2°C in room air, Tr did not increased in 35%O2, and was 31.8±0.4°C in 10%O2. VO2 was high (P7=8.6±0.8; P15=9.4±0.4 ml/min/100g) compared to sea level rats (5 ml/min/100g in 20g P10 rats). In P7 and P15 rats, VO2 increased in 35%O2 and dropped in 10%O2. Our results suggest that chronic hypoxia delays the establishment of thermoregulation and increases metabolic rate in newborn rats. The drop of Tr and VO2 in 10%O2 were however well maintained. Founded by NSERC