bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenylethyl isothiocyanate (PITC) inhibits lipopolysaccharide (LPS)‐stimulated inflammatory responses in Raw 264.7 murine macrophages
Authors: Lee yeo myeong , Seon Mi Ra , Cho Han Jin , Song Hye Rim , Kim Jong‐Sang , Park Jung Han Yoon
Published: June 2021

Phenylethyl isothiocyanate is the hydrolysis product of the glucosinolate gluconasturtiin present in Brassica vegetables which have been linked to decreased cancer incidence in epidemiological studies. Because recurrent or chronic inflammation has been implicated in the development of a variety of cancer, the present study assessed the effects of the PITC on LPS‐stimulated inflammatory responses in Raw 264.7 cells. Treatment of cells with increasing concentrations (0.5 ‐ 5 μM) of PITC resulted in a dose‐dependent decrease in nitric oxide (NO) and prostagrandin E2 (PGE2) production. Western blot analysis and RT‐PCR analysis revealed that PITC dose‐dependently inhibited LPS‐induced expression of the iNOS protein and mRNA, respectively. In addition, PITC suppressed LPS‐induced translocation of the p65 subunit of NF‐κB to the nucleus, and degradation of inhibitor of κB. Furthermore, treatment of Raw 264.7 cells with LPS dramatically activated Akt, ERK1/2, SAPK/JNK, and p38 MAP kinase. PITC pretreatment significantly inhibited LPS‐induced phosphorylation of ERK1/2 and Akt, whereas the phosphorylation of other MAPK family proteins, SAPK/JNK or p38, was not affected by PITC pretreatment. The present data indicate that PITC exhibits anti‐inflammatory properties via the inhibition the NF‐κB signaling pathway.
Energy restriction with high‐protein diets decreases visceral fat mass but not fasting and postprandial inflammation in overweight insulin‐resistant rats
Authors: Mariotti Francois , Xu Liping , Mathé Véronique , Brunsard Julie , Magné Joelle , Bos Cécile , Tomé Daniel , Gaudichon Claire , Huneau Jean‐Francois
Published: June 2021

Visceral obesity may worsen the low‐grade postprandial inflammatory response to a high‐fat meal. To test this hypothesis, we studied the effects of energy restriction in rats with diet‐induced overweight. Overweight rats were given free‐access to a high‐fat diet (HF), or restricted to 60% of the spontaneous intake of HF rats, with either the same diet (HFR) or protein‐rich diets (HPR) for 5 weeks. Before and at the end of this period, rats were administered a high‐fat meal and plasma insulin, triglycerides and cytokines were monitored after meal. Rats were killed at the end of the experiment and the epididymal fat pads were weighed.

At the end of the experiment, the weights of the restricted rat were similar between HFR and HPR groups and 19%‐lower than in HF rats. However, the epididymal fat masses were slightly but significantly lower in HPR rats than in HFR and HF rats (14.5±0.6, 17.4±1.2 and 24.0±2.3 g, respectively). Fasting insulin did not change in restricted rats, while almost doubling in HF rats. At the end of the experiment, fasting and postprandial increases in IL‐1β, IL‐6, MCP‐1 and PAI‐1 did not differ between groups. Altogether, these results suggest that decreasing visceral adiposity in insulin‐resistant overweight rats does not impact fasting and postprandial low‐grade inflammation.
Blood‐flow fluctuation during light‐resistance exercise may enhance the effectiveness of high‐protein snack on the increase of muscle mass and strength
Authors: Suzuki Masashige , Kato Yuushi , Sawada Atsushi , Numao Shigeharu
Published: June 2021

We have demonstrated the preventive effects of a high‐protein snack 3hrs after a basal meal

under voluntary tower climbing exercise on the loss of muscle and bone mass in aging‐model rats receiving glucocorticoid injections. However, this "missile nutrition" effects of the snack could no be observed without exercise. In this study we examined chronic effects of light‐resistance exercise (15‐min dumbbell exercise using 300g brown‐rice filled dmbbells) after a high‐protein (15g)‐sucrose (15g) snack (HPS) on the forearm and thigh muscle mass and hand‐grip and leg‐extension strength, comparing with that of exercise or HPS alone in young adults. Neither dumbbell exercise nor HPS alone increased muscle mass and strength, excepting a significant increase of hand‐grip strength with exercise. Contrarily, both muscle mass and strength in either forearm or thigh muscle was significantly increased and body fat mass was significantly decreased by the intake of HPS followed by dumbbell exercise 45 min after HPS ingestion. The dumbbell exercise fluctuated greately the blood flow in the muscle tisues and lowered the peaked plasma amino acid concentrations after HPS, suggesting an enhanced amino acid uptake by the muscle to effective protein synthesis.
Effect of three supplements with equal micronutrient content on serum zinc concentrations in Mexican children
Authors: García‐Guerra Armando , Rivera‐Dommarco Juan , Neufeld Lynnette , Domínguez‐Islas Clara P.
Published: June 2021

Objective

To evaluate the efficacy of three nutritional supplements with equal micronutrient content on serum zinc in children. Methods: 54 communities were randomly assigned to receive three sources of multiple micronutrients: a fortified food (FF), syrup (SY) or Sprinkles (SK), all three containing 10 mg of Zinc. Children 6 to 12 mo of age, beneficiaries of the Oportunidades program, were recruited and supplemented to 24 mo age. Supplements were delivered daily and compliance recorded. Serum zinc was measured at baseline and after 4 mo supplementation on a non‐random sub‐sample.

Results

At baseline serum zinc did not differ between groups (FF: 74.90±14.6; SY: 70.96±13.8; SK:72.73±14.6 μg/dL). After 4 mo of supplementation, serum zinc ( μg/dL) was significantly higher (p<0.01) in the SY (104.80±35.3) than SK (93.57±24.9) and FF (81.29±17.6). A dose response in serum zinc (µg/dL per dose) was found for SY (0.39, p<0.01) and SK (0.18, p=0.03), with no significant differences by baseline serum zinc status. A similar but non significant (p=0.07) trend was found with FF, but only among children who were zinc‐deficient at baseline (0.24, p=0.065). Serum zinc increment was negatively associated with serum zinc concentration at baseline in the three groups.

Conclusion

Syrup and Sprinkles were more efficacious than the fortified milk‐based food Nutrisano to improve zinc status in children.
Pharmacological Probes for AMPA Receptors
Authors: England Pamela M.
Published: June 2021

Alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isooxazolepropionic acid (AMPA) receptors are a family of glutamate‐gated ion channels that mediate the majority of fast, excitatory synaptic transmission in the mammalian central nervous system. In addition, changes in the efficacy of AMPA receptor mediated synaptic responses underlie many forms of normal plasticity (e.g. long‐term potentiation and memory). Moreover, AMPA receptors have been implicated in several neuropathologies (e.g. schizophrenia and Alzheimer's disease). Changes in the expression pattern of AMPA receptors may provide a molecular basis for both normal and maladaptive forms of plasticity. Data describing the development and use of novel pharmacological tools for studying the activity of AMPA receptors will be presented.
Effects of DTPA on zinc flux in leukemia cell lines
Authors: Sankavaram Kavitha , Grattan Bruce J , Freake Hedley C
Published: June 2021

Previous studies from our laboratory with Zn‐65 have shown that H4IIE (rat hepatoma) and GH3 (rat pituitary tumor) cell lines reduce zinc efflux in the presence of the extracellular metal chelator DTPA (diethylenetriaminepentaacetic acid). Primary rat hepatocytes and anterior pituitary cells give out more zinc under the same conditions. This may reflect the greater zinc requirements of the rapidly dividing transformed cells. Here, we examined the effects of DTPA on radioactive Zn‐65 flux in C58(NT) (rat thymic lymphoma) and HL‐60 (human promyelocytic leukemia) cell lines. When these cells were co‐incubated with DTPA (50 µM) and Zn‐65, uptake of label was reduced. But when cells were pre‐labeled with Zn‐65 and then incubated with DTPA, the chelator increased efflux of Zn‐65. Despite being transformed, these cell lines behaved similarly to primary cells. Since the leukemia cells grow in suspension unlike the monolayer cell lines previously examined, we determined whether non‐adherence contributed to the difference in response to zinc deprivation. Experiments were repeated with H4IIE cells kept in suspension by growth in roller bottles. However, even under these conditions, DTPA enhanced retention of cellular zinc in the hepatoma cells. Thus, leukemia cells differ from other transformed cell lines in their homeostatic response to zinc deprivation and this is not due to their inability to form a monolayer.
The effect of hyperbaric oxygen (HBO2) on the anticancer effect of artemesinin (ART) on Molt‐4 human leukemia cells
Authors: Ohgami Yusuke , Chung Eunhee , Lai Henry C , Quock Raymond M , Elstad Catherine A
Published: June 2021

ART reacts with free iron to form cytotoxic free radicals. It has been shown to be selective in killing cancer cells compared to its effect on normal cells because cancer cells contain more intracellular free iron. Furthermore, there is evidence in the literature that HBO2‐100% oxygen administered at elevated atmospheric pressure (ATA)‐may be beneficial in the treatment of cancer. The hypothesis of the present study is that the anticancer activity of ART can be enhanced by exposure to HBO2. In our studies, after pretreatment with 12 µM human holotransferrin, 10 µM ART was added to the culture and Molt‐4 cells (3.2 × 105 cells/mL) were exposed for 90 min to one of three different conditions: control (37ºC, 5% CO2 / 95% RA, normal ATA); room air [room temperature (RT), RA, normal ATA]; and HBO2 (RT, 100 % O2, 2.5 ATA). Cell growth and viability were determined at 1, 2, 4, 6, 12, 24 and 48 hrs. Differences in growth among treatments were noted after 6 hr of incubation. After 48 hr of incubation, growth of cells exposed to ART alone or HBO2 alone was 85.3% of that of cells grown under ART‐free control conditions. Combined exposure to ART and HBO2 resulted in an additional 22.1% decrease in growth. These findings suggest the possibility that combined HBO2 and ART exposure can be developed into an effective chemotherapeutic strategy for cancer treatment. (Supported by the Washington State University College of Pharmacy.)
Potential role of the toll‐like receptor signaling pathway in equine laminitis
Authors: Stokes Ashley Michelle , McGeachy Matthew , Carlisle Katherine , Garza Frank , Keowen Michael L
Published: June 2021

Equine laminitis, a very common and painful disease of the laminar tissues of the foot, often results from conditions such as GI disruption (colic) or metabolic disease (Cushing's disease). Identification of the link between these primary diseases and the subsequent failure of the tissues of the foot is lacking and discovery would allow targeted development of therapeutics.

Our objectives were to design equine primers/probes to evaluate key mediators of the Toll‐Like Receptor (TLR) Signaling Pathway to determine its role in laminitis. Specifically we examined TLR4, cluster of differentiation 14 (CD‐14), TLR2, phosphatidylinositol‐3‐kinase regulatory subunit 1 (PIK3R1), inhibitor of NF‐¿B (INFKB) and cyclooxygenase 2 (COX2).

Primers and probes were designed from cloned equine samples, predicted sequences, and multi‐species compilations and were validated. Equine liver samples (normal n=6, laminitic n=15, clinical signs of metabolic disease n=4) were used in TaqMan RT‐PCR.

Laminitic horses consistently upregulated these genes and metabolic cases (highly correlated with laminitis development) demonstrated significant (p<0.05) upregulation of CD14, TLR2, PIK3R1 and INFKB. These findings demonstrate key mediators of the TLR signaling pathway deserve further evaluation to determine their role in the pathogenesis of laminitis.

Funding provided by the LSU Equine Health Studies Program.
Albuminuria and Renal Histopathology in Hyperlipidemic, Insulin Resistant Swine
Authors: Merricks Elizabeth , Bellinger Dwight , Clemmons David , Jennette Charles , Raymer Robin , Nichols Timothy
Published: June 2021

Human diabetic nephropathy is characterized by progressive renal insufficiency, albuminuria, and characteristic histopathology. Both insulin resistance (IR) without overt diabetes as well as hyperglycemia in diabetes are risk factors for chronic renal disease in humans.

Objective

To determine the extent of renal dysfunction and histopathology in hypercholesterolemic IR pigs and if these changes mirror the nephropathy seen in IR humans.

Methods

18 adult IR pigs were fed a high fat diet and 6 adult FH insulin sensitive (IS) pigs were fed a low fat diet for 12 months. Urine was analyzed for albumin/creatinine ratio (UA/C) and kidneys for histopathology.

Results

All pigs had comparable hypercholesterolemia. One IR pig died with renal failure after 3 months on diet (serum creatinine 9.4 mg/dl). No other pig had an increase in serum creatinine. 9/18 IR pigs that completed 12 months of diet had UA/C >30 and only these 9 had higher blood pressure and renal histopathology: glomerulosclerosis, increased mesangial matrix area, tubulointerstitial fibrosis, and arteriolar hyalinosis (p < 0.05). 0/6 IS FH pigs had a UA/C> 30 or renal histopathology.

Conclusion

Renal histopathology in IR pigs appears to mirror the phenotype of a diabetic nephropathy when albuminuria is present. These data also suggest that IR is necessary but insufficient for nephropathogenesis in pigs. Research support: NCBC 2008‐MRG‐1101, R01 HL069364.
Shear stress induces actin rearrangement in endothelial cells
Authors: Zhang Zhenxian , AbouAlaiwi Wissam A , Nauli Surya M
Published: June 2021

Background

It has been known that endothelial cells can respond to fluid‐shear stress. Recently, our laboratory and others have shown that cilia are mechanosensory organelles that allow endothelial cells to respond to shear stress‐induce changes in cytosolic calcium, gene expression and nitric oxide production. We now show that cilia are also involved in shear‐induced actin rearrangement in endothelial cells.

Methods and Results

Wild‐type (WT) and cilialess mutant (Tg737orpk) endothelial cells have different responses to shear stress. We show here for the first time that unlike WT cells, Tg737orpk cells do not respond to shear‐induced actin rearrangement. WT cells are very sensitive to shear stress; shear induced with phosphate buffer solution is sufficient to cause actin rearrangement in WT cells. Expression level of glycogen synthase kinase‐3β (GSK‐3β) does not seem to be regulated by shear‐induced actin rearrangement. Differential shear‐induced time courses on GSK‐3β expression are not statistically different between WT and Tg737orpk cells.

Conclusion

Shear stress‐induced actin rearrangement in WT and Tg737orpk endothelial cells is regulated differently. Cilia may play a significant role in shear‐induced actin rearrangement.
Donepezil differentially interacts with gonadal hormones in male rats responding under a multiple schedule of repeated acquisition and performance
Authors: Leonard Stuart T. , Hearn John K. , Winsauer Peter J.
Published: June 2021

Gonadal steroid hormones can mediate learning and memory, presumably by enhancing the functional status of cholinergic neurons. In contrast to the effects of estrogen in female rats, some evidence suggests that testosterone in male rats may decrease acetylcholine levels, and thus, the activity of the cholinergic system. The present study investigated the interactive effects of testosterone and donepezil, an acetylcholinesterase inhibitor, in subjects responding under a multiple schedule of repeated acquisition and performance of response chains. Donepezil dose‐effect curves (0.56‐10 mg/kg) were determined in males that were gonadally intact, gonadectomized, gonadectomized with testosterone replacement, or gonadectomized with estradiol replacement. For all four groups, donepezil produced dose‐dependent rate‐decreasing and error‐increasing effects in both the acquisition and performance components. Gonadally intact, GX, and GX+T males showed less disruption of response rate in both the acquisition and performance components than GX+E males. In terms of accuracy, GX+E males had the highest percentage of mean errors after control injections and also after donepezil administration in both the acquisition and performance components. Together, these data demonstrate that donepezil differentially interacts with gonadal hormones, and estrogen in particular, in male rats. Supported by DA 019625.
Hypothalamic disinhibition stimulates retrotrapezoid nucleus (RTN) chemosensitive neurons in vivo
Authors: Fortuna Michal G , Stornetta Ruth L , West Gavin H , Guyenet Patrice G
Published: June 2021

Stimulation of neurons within the dorsomedial hypothalamic nucleus (DMH) and perifornical area (PeF) produces an increase of respiratory activity, tachycardia and a marked pressor response. The descending pathway mediating the respiratory arm of this complex response is not well characterized. Intrinsically pH sensitive retrotrapezoid nucleus (RTN) neurons are proposed to produce excitatory drive to the respiratory network (CPG). We sought to determine if pharmacological stimulation of DMH/PeF would result in an increased activity of RTN neurons, and to determine if this response is dependent on the CPG. RTN unit activity was monitored in adult, isoflurane‐anesthetized rats. Here we report that stimulation of DMH/PeF with the selective GABAA antagonist, gabazine, increases phrenic nerve activity (PNA) as previously reported and markedly increases the discharge rate of RTN units. This effect was independent of the CO2 level and respiratory modulation of the RTN cells in general was not affected. Moreover after silencing the CPG with morphine, hypothalamic stimulation was still able to increase RTN activity, even before the CPG was reactivated. Morphine also had a mild inhibitory effect on the RTN neurons. In summary we propose that the descending pathway from DMH/PeF that activates respiration might be in part mediated through RTN chemosensitive neurons.
Pharmacological characterization of the alpha 2‐adrenergic receptor inhibiting rat hippocampal CA3 epileptiform activity: comparison of ligand efficacy and potency
Authors: Xu Ke , Nelson Brian , Goldenstein Brianna , Pribula Jacquline , Luger Elizabeth , O'Shea Lorraine , Boese Sarah , Jurgens Chris , Porter James , Doze Van
Published: June 2021

Pharmacological characteristics of the α2 adrenergic receptor (AR) that inhibits epileptiform activity in the hippocampal CA3 region were studied in rat brain slices. The effects of a large number of αAR antagonists on epinephrine (EPI)‐mediated inhibition of epileptiform activity were scrutinized with Schild plot analysis and compared to binding values for the rat and human α1, α2, 5‐HT1A, D2, I1, and I2 receptor subtypes. A perfect correlation and slope was found for the rat α2AAR, but not the other receptors, indicating that α2AARs solely mediate this effect. The actions of different chemical classes of α2AAR agonists at inhibiting epileptiform activity were then determined. While the imidazolines and guanidines exhibited the highest potency, the catecholamines had greater intrinsic activity compared to norepinephrine (NE). Dexmedetomidine, an imidazoline and guanabenz, a guanidine, were the most potent. In contrast, the catecholamines EPI and α‐methyl‐NE were the most efficacious. Furthermore, the α2AAR‐mediated inhibitory actions of EPI on hippocampal CA3 epileptiform burst frequencies were neither sex nor age‐dependent. These findings suggest that the activation of α2AARs could provide a new pharmacotherapeutic strategy for treating epilepsy and highlight the need for selective α2A agonists. Supported by ND EPSCoR, NSF CAREER 0347259, NSF REU Site 0639227, NIH P20 RR016741, APS and ASPET SURF.
5‐HT7 Serotonin Receptor Increases Neurite Complexity of Hippocampal Neurons Through Novel PKA‐LIM Kinase 1 Dependent Mechanism
Authors: Krbanjevic Aleksandar , Liu Guoquan , Strekalova Elena , Profirovic Jasmina , Voyno‐Yasenetskaya Tatyana
Published: June 2021

Serotonin [5‐hydroxytryptamine (5‐HT)] containing neurons are critical in memory formation. Previously, we found that 5‐HT7R promotes neurite outgrowth in PC12 cells and dissociated hippocampal neurons. A serine/threonine kinase, LIM kinase 1 (LIMK1) is a novel cytoskeleton regulator that upon phosphorylation leads to increase in actin filament stability and promotion of neurite outgrowth. The purpose of this study is to determine whether genetic deletion of LIM kinase 1 (LIMK1) could suppress 5‐HT7R induced increase in neurite complexity in hippocampal neurons.

Using Western blot analysis, we found that 5‐HT7R can induce phosphorylation of LIMK1 in PC12 cells. 5‐HT7R can activate adenylate cyclase and increase intracellular levels of cAMP. Stimulation of PC12 cells with forskolin induced adenylate cyclase (AC) mediated LIMK1 phosphorylation. Furthermore, pretreatment of 5‐HT7R‐transfected PC12 cells with PKA specific inhibitor reduced LIMK1 phosphorylation. These findings suggest that 5‐HT7R‐AC‐cAMP‐PKA could be a novel pathway that leads to LIMK1 phosphorylation. We found that 5‐HT7R protein is expressed in pyramidal neurons of CA3 hippocampal region. To further explore importance of 5‐HT7R‐LIMK1 phosphorylation in neurons, we will determine morphology of 5‐HT7R‐expressing CA3 neurons in organotypic slices of wild type and LIMK1 ‐/‐ mice.

This project is supported by NIH R01 grant.
Effect of phosphodiesterase 5 inhibition on systemic hemodynamics and regional blood flows in exercising swine
Authors: Bender Shawn B , de Beer Vincent J , Merkus Daphne , Duncker Dirk J , Laughlin M. Harold
Published: June 2021

Phosphodiesterase 5 (PDE5) inhibitors have garnered wide therapeutic use; however, the effect of PDE5 inhibition on blood flow to skeletal muscle, which expresses PDE5, during exercise in healthy animals has not been determined. We therefore investigated the effect of PDE5 inhibition on exercise‐induced vasodilation in vivo with regional blood flows determined using microsphere infusion at rest and during acute exercise. Chronically instrumented swine (n=5) were exercised on a treadmill (0‐5 mph) and systemic hemodynamics and regional blood flows were compared before and during infusion of the PDE5 inhibitor EMD360527 (0.3 mg/kg/min iv). At rest, inhibition of PDE5 had no effect on mean aortic pressure whereas heart rate and cardiac output increased and systemic vascular resistance (SVR) decreased by 17% consistent with peripheral vasodilation. Visceral, cardiac, cerebral and skeletal muscle blood flows were unchanged. The PDE5‐induced reduction in SVR was maintained during acute exercise at 4 mph and blood flows to the superficial portions of the triceps brachii long head and vastus lateralis in the forelimb and hindlimb, respectively, were increased. All other regional blood flows were unchanged. Thus, inhibition of PDE5 induces peripheral vasodilation and preferentially increases skeletal muscle blood flow during acute exercise in swine. Supported by NIH and the Dutch Heart Foundation.
Ca2+‐dependent K+‐channels (IK1, SK3) mediate arteriolar dilation in a distinct stimulus‐dependent fashion
Authors: de Wit Cor , Milkau Malte , Schmidt Volker J , Wolfle Stephanie E , Hoyer Joachim , Kohler Ralf
Published: June 2021

Endothelial hyperpolarisation is an important mechanism in the regulation of vascular tone. Herein, the Ca2+‐dependent K+‐channels SK3 (KCa2.3) and IK1 (KCa3.1) have been proposed to take center stage, however, their precise role in endothelium‐dependent dilation in response to agonists or active hyperemia is not clearly understood. We studied microvascular dilations in mice deficient for only IK1 (IK‐ko) or IK1 and SK3 (IKSK‐ko) in response to agonist application as well as to skeletal muscle contraction by intravital microscopy.

After blockade of prostaglandins and NO, acetylcholine (ACh)‐induced dilations were significantly reduced in IK‐ko mice (at 10 µM from 67 to 49%) and further reduced to 25% in IKSK‐ko. However, blockade of SK3 by UCL1684 in wildtype did not attenuate the ACh‐response. Dilations elicited by adenosine or SNP remained mostly unaffected by deficiency or blockade of KCa. Skeletal muscle contraction initiated by stimulation of the motor nerve dilated arterioles gradually depending on time and frequency. The maximal dilation after 60 s stimulation was attenuated by blockade of SK3 in wt (UCL: from 74 to 37%). In contrast, dilation was fully intact in IK‐KO (89%), but also reduced in IKSK‐ko (50%).

We conclude that IK1 is mainly responsible for endothelial hyperpolarisation and dilation upon ACh, whereas SK3 only contributes in the absence of IK1. In contrast, exercise hyperemia relies heavily on SK3‐activity and IK1 seems to be of minor importance. These data suggest distinct roles of IK1 and SK3 in arteriolar dilation and may involve specific activation pathways which depend on the stimulus.
Hyaluronidase treatment of the glycocalyx attenuates insulin sensitivity during an intravenous glucose tolerance test in rats
Authors: Eskens Bart , Cobelens Hanneke , Vink Hans , VanTeeffelen Jurgen
Published: June 2021

The potential of insulin to stimulate glucose uptake from blood to tissues is closely related to the ability of insulin to increase capillary blood volume. The endothelial glycocalyx forms a relatively thick interface between blood and the vascular wall, and recent studies have demonstrated that recruitment of glycocalyx is a powerful tool to effectively increase capillary blood volume. We therefore hypothesized that enzymatic degradation of the glycocalyx impairs the potential of insulin to stimulate glucose disposal.

Blood glucose levels and plasma insulin levels were determined following an i.v. bolus of glucose (0.5g/kg; IVGTT) and the area under the curve for glucose (Aglu, in mg/dl*90 min), and for insulin (Ains mU/ml*90 min) were calculated.

The Aglu did not differ between the first and second IVGTT following a bolus of saline (3504±821 (mean±SD) versus 3823±1074), nor the Ains (1248±437 versus 1424±377). In contrast, 60 minutes following a bolus of hyaluronidase (500 U) Ains increased significantly (1128±646 versus 1818±772 (p<0.05)), without a change in Aglu (3171±539 versus 3090±681).

In conclusion, enzymatic degradation of the glycocalyx resulted in a 60% increase in plasma insulin required for normal glucose disposal. This finding is consistent with an important contribution of the glycocalyx to the ability of insulin to stimulate glucose uptake.

Supported by DFN 2006.00.027 and NHF 2005T037
VEGF Regulates Gene Expression of Matrix Factors During Cervical Remodeling in Rodents
Authors: Hughes Stephanie Cristina , Hughes Stacey Cristina , Mowa Chishimba Nathan
Published: June 2021

Cervical remodeling is a physiological inflammatory process regulated by various factors including vascular factors. We have previously shown that VEGF regulates multiple genes involved in cervical remodeling using DNA microarray analysis, such as growth factors, inflammatory factors, neuronal factors and matrix proteins. Among the matrix factors regulated by VEGF are biglycan, collagen type I, II, IV and V, decorin, fibronectin and others. Here, we validate the DNA microarray data using real time PCR and found that in the gene expression of a selected number of genes were down‐regulated such as decorin. We conclude that VEGF plays a role in cervical remodeling by regulating various genes including matrix factors.
Role of insulin resistance in heart failure and high fat diet
Authors: Christopher Bridgette A , McElfresh Tracy A , Chen Xiaoquin Q , Croniger Colleen M , Chandler Margaret P
Published: June 2021

Both heart failure (HF) and high saturated fat diets (SAT) are associated with the development of insulin resistance. Whether insulin resistance exists in a model that involves both HF+SAT has not been studied. Previous studies reported that HF+SAT rats have improved left ventricular contractile and mitochondrial function compared to HF animals fed normal chow (NC). We hypothesize that myocardial insulin resistance exists in HF+SAT rats and that it serves a cardioprotective role. Male Wistar rats underwent coronary ligation or sham surgery and were fed NC (10% kcal fat) or SAT (60% kcal saturated fat) for 8 weeks. Five weeks post ligation, a glucose tolerance test (GTT) was performed to determine peripheral insulin resistance via plasma glucose and insulin concentrations. Echocardiography was done at 7 weeks post ligation to assess cardiac function. Tissue was harvested 8 weeks post ligation to assess the insulin signaling pathway via targets such as AKT, mTOR, GSK3β, eIF‐2, and AMPK by protein and RNA analysis. Preliminary results indicate that HF+NC and Sham+SAT have abnormal GTTs compared to Sham+NC. However, HF+SAT animals do not have impaired GTT. Ongoing studies will assess whether differences in GTT result from alterations in insulin signaling and whether these alterations in HF+SAT serve in a cardioprotective function in heart failure. (Grant # NHLBI HL‐081857 & AHA 0535361N)
Mitochondrial uncoupling and remodeling during caloric restriction: Implications for oxidative stress and aging
Authors: Estey Carmen , Seifert Erin L , Harper Mary‐Ellen
Published: June 2021

Caloric restriction (CR) mitigates aging; however, mechanisms are poorly understood. The 'uncoupling to survive' theory suggests that mitochondrial uncoupling protects cells from reactive oxygen species (ROS). It is unclear how uncoupling protein 3 (UCP3) in skeletal muscle is involved.

Objective

To characterize the effects of 2 wk 40% CR in wildtype (Wt) and UCP3 transgenic (UCP3 Tg) mice.

Hypothesis

In an already uncoupled system, CR provides no further ROS protection.

Approaches

Muscle mitochondria and whole body assessments of UCP3's contribution to uncoupling and oxidative stress.

Results

In ad libitum fed mice, UCP3 Tg weighed less (p < 0.005; n = 18), had less muscle (p < 0.005; n = 8‐9), expended ~5% more energy and burned more carbohydrate (n = 9) than Wt. UCP3 Tg mitochondria produced less ROS than Wt (p < 0.05; n = 4‐5). CR in Wt mice did not decrease ROS production and increased it in UCP3 Tg (p < 0.05). CR tended to increase UCP3 and ANT in Wt and decrease ANT in UCP3 Tg (n = 3). MnSOD was unchanged in both genotypes, although UCP3 Tg had less MnSOD vs. Wt (p < 0.005; n = 3). Oxidative damage (4‐hydroxynonenal adducts) was unchanged in both genotypes (n = 3). However, 1 mo CR decreased ROS production in Wt, but not in UCP3 Tg mice (n = 2‐4). In summary, 2 wk CR is insufficient in mice, unlike rats (Bevilacqua et al, 2004), to mitigate oxidative stress; 1 mo is effective. Surprisingly, ROS production increases in UCP3 Tg mitochondria with 2 wk CR. Support: NSERC of Canada.
Oral Contraceptive Use Impairs Muscle Gains in Young Women
Authors: Lee Chang Woock , Newman Mark A. , Riechman Steven E.
Published: June 2021

Background

Many active young women use oral contraceptive (OC) yet its effect on their body composition and exercise performance has not been thoroughly studied. We examined the effects of oral contraceptive on muscle responses to resistance exercise training (RET).

Methods

2 groups of 18‐31 year old subjects (OC: n=34, no OC: n= 39) underwent 10 week whole‐body RET (3x/week, 3 sets, 6‐10 reps, at 75% of max strength). Body composition was determined using hydrostatic weighing. Blood samples were taken before/after the training and assayed for DHEA, DHEAS, cortisol, and IGF1.

Results

There were significant differences in lean mass gains (OC: 2.1±2.1%, no OC: 3.5±3.2% / OC: 1.0±1.0kg, no OC: 1.6±1.4kg, p<0.05). Plasma concentrations of DHEA, DHEAS, and IGF1 were significantly lower and cortisol was elevated in OC (DHEA: OC 9.5±4.4, no OC 13.6±4.6 / DHEAS: OC 1451±685, no OC 2196±1008 / IGF1: OC 163±42, no OC 239±126 / cortisol: OC 33.4±10.8, no OC 24.3±9.4, ng/ml, p<0.05). There also were significant differences in lean mass gains depending on androgenicity of the progestin in OC (low=2.5%, medium/high=0.3%, P<0.05).

Conclusion

Oral contraceptive use impaired muscle gains in young women which was associated with lower DHEA, DHEAS and IGF1 and higher cortisol. The diminished lean mass gain may be related to the androgenicity of progestin which may bind to androgen receptor and inhibit its function.
Role of RVLM in mediating sympathetic nerve responses to hyperthermia
Authors: Hosking Kimberley , Fels Richard , Kenney Michael J
Published: June 2021

Hyperthermia produces profound increases in visceral sympathetic nerve discharge (SND) in young rats. The brainstem in rats contains the essential neural circuitry required for mediating sympathoexcitatory responses to heating, however, specific brainstem sites involved in mediating SND responses to hyperthermia are unknown. Because the rostral ventral lateral medulla (RVLM) plays a critical role in the maintenance of basal SND and in mediating cardiovascular and sympathetic responses evoked from brainstem sites, we tested the hypothesis that inhibition of RVLM synaptic transmission would alter SND responses to acute heating in chloralose/urethane‐anesthetized young SD rats. Renal and splenic SND was recorded in baroreceptor‐intact and ‐denervated rats before and during heating (core temperature, Tc, increased to 41.5°C). SND was increased more than 100% from control during heating. With Tc maintained at 41.5°C, bilateral RVLM microinjections of muscimol (400 and 800 pmol/40 nl) decreased SND to 30% below control values within one minute. Similar results were produced by RVLM lidocaine (40 nmol/40 nl) microinjections. RVLM microinjections of muscimol and lidocaine in rats at a Tc of 38°C produced similar decreases in SND. These results demonstrate that visceral sympathoexcitatory responses to hyperthermia in anesthetized rats are dependent on RVLM neural circuits.
Subunit stoichiometry of the endogenous endothelial ISOC channel in the pulmonary microcirculation
Authors: Cioffi Donna L , Uhlig Stefan , Stevens Troy
Published: June 2021

The endothelial ISOC channel is a calcium selective store‐operated calcium entry channel whose activation is an important step leading to intercellular gap formation and increased permeability. The transient receptor potential canonical (TRPC) proteins 1 and 4 contribute to ISOC channel structure. We have recently determined that the subunit stoichiometry of the ISOC channel in rat pulmonary artery endothelial cells is one TRPC1 plus three TRPC4 subunits. While we know that rat pulmonary microvascular endothelial cells also possess ISOC, it is unclear as to whether the microvascular ISOC channel has the same molecular architecture as that of the pulmonary artery. We therefore sought to determine the subunit stoichiometry of the pulmonary microvascular endothelial ISOC channel under cultured and native conditions. Here we used cultured pulmonary microvascular endothelial cells as well as caveolae‐rich membrane fractions from the isolated perfused rat lung. TRPC1 and TRPC4 subunits were tagged with fluorophore (Cy3 and Cy5) labeled antibodies and Förster Resonance Energy Transfer (FRET) experiments performed. FRET was observed between TRPC1‐Cy3 and TRPC4‐Cy5 as well as TRPC4‐Cy3 and TRPC4‐Cy5, but not between TRPC1‐Cy3 and TRPC1‐Cy5. Collectively these data indicate that the pulmonary microvascular ISOC channel contains one TRPC1 plus three TRPC4 subunits. Supported by HL60024 and F32HL086124.
Estrogen replacement enhances EDHF‐mediated vasodilation of mesenteric and uterine resistance arteries: role of endothelial cell Ca2+
Authors: Gokina Natalia I , Burger Natalie Z , Osol George , Kuzina Olga Y
Published: June 2021

Endothelial‐derived hyperpolarizing factor (EDHF) plays an important role in the regulation of vascular microcirculatory tone. This study aimed to explore the role of estrogen in the control of EDHF‐mediated responses in reproductive and non‐reproductive vasculature, and to study the contribution of endothelial cell (EC) Ca2+ signaling to this process. Mature female rats (n = 63) underwent ovariectomy, with one‐half receiving 17β‐estradiol replacement and the other half serving as estrogen‐deficient controls. Uterine or mesenteric resistance arteries were cannulated, pressurized, and loaded with fura‐2 for measurements of EC [Ca2+]i. NO and prostacyclin production were inhibited with 200 µM LNNA and 10 µM indomethacin, respectively. ACh effectively dilated arteries pre‐constricted with phenylephrine, but failed to induce dilatation of vessels pre‐constricted with depolarizing K+ solution. Estrogen replacement increased arterial sensitivity (EC50) to ACh five‐ and two‐fold in uterine and mesenteric arteries, respectively. Estrogen replacement was also associated with both increased basal and ACh‐stimulated elevations of EC [Ca2+]i in uterine, but not mesenteric vessels. These data demonstrate that estrogen promotes EDHF‐related mechanism(s) more effectively in reproductive vs. non‐reproductive vessels, in part through enhanced EC Ca2+ signaling.

Supported by NIH HL067250 and HL073895
Molecular evidence for multiple purinergic P2X receptor (P2XR) subtypes in supraoptic nucleus (SON)
Authors: Sladek Celia D. , Stevens Wanida , Lawson Regan R. , Song Zhilin
Published: June 2021

Pharmacologic approaches provided evidence for involvement of P2X2, 2/3, and 7R subtypes in ATP evoked calcium signaling in SON neurons and neurohypophyseal vasopressin and oxytocin (VP/OT) release. Since desensitization of these subtypes ranges from rapid (<100msec for P2X3), to slow (within 1 min for P2X2), to non‐desensitizing (P2X2/3 heteromers and P2X7), the subtypes expressed could dictate whether ATP induces an acute response or one that lasts for hours. Using Western Blot (WB), co‐immunoprecipitation (co‐IP), and immunohistochemistry (IHC), we provide evidence for these receptors in SON. IHC for P2X2 and P2X3 demonstrated intense labeling of SON neurons that was absent with peptide absorption. On WB, the same P2X2 antibody recognized a protein the appropriate size for P2X2R (~57kD) in microdissected rat SON. Co‐IP demonstrated that a portion of P2X2R exists as a heteromer with P2X3. WB analysis of P2X7R‐ir demonstrated the presence of ~70kD protein in SON from rat and wild type mouse that is absent in P2X7 knockout mouse. These findings provide molecular evidence to substantiate the functional involvement of P2X2, and the non‐desensitizing P2X2/3 and 7R subtypes in VP/OT release. Functional recruitment of the non‐desensitizing subtypes may underlie sustained release of VP during dehydration and may be important in preventing hemorrhagic and septic shock. Supported by AHA GIA #0850149Z.
Role of adrenal medulla in dexamethasone‐induced hypertension
Authors: Soto‐Pina Alexandra Estela , Kadapakkam Sheela , Mehring Cynthia , Hinojosa‐Laborde Carmen , Strong Randy
Published: June 2021

Dexamethasone (DEX) was reported to induce hypertension and increase expression of tyrosine hydroxylase (TH), the rate limiting enzymatic step in catecholamine synthesis, in the adrenal medulla. We hypothesized that: 1) DEX increases TH message in the adrenal medulla, and 2) removal of the adrenal medulla attenuates DEX‐induced hypertension. Fisher 344 rats were implanted with radio telemetry transmitters to record blood pressure and heart rate. After 5 days of baseline recording, rats were divided into 2 groups: SHAM and Adrenal medullectomy (ADMX) surgeries. Both groups received vehicle (VEH) or DEX in drinking water (0.1 mg/day for 7 days), starting 5 days after surgery. Adrenal medullas were collected from the SHAM‐VEH and SHAM‐DEX groups and RNA was isolated to quantify TH mRNA by qPCR. The results showed that TH message in DEX treated adrenal medullas increased by 5 fold. DEX increased blood pressure in SHAM‐DEX by 17± 2 mmHg and in ADMX‐DEX by 18±1 mmHg. The results of the study showed that DEX increased TH message in the adrenal medulla, but DEX‐induced hypertension was not prevented by ADMX. We conclude that DEX‐induced hypertension is not completely dependent on the adrenal medulla. Future studies will focus on the contribution of other sympathetic ganglia to DEX‐induced hypertension. Support provided by: Department of Veterans Affairs and CONACYT.
Astrovirus Induces Diarrhea in Turkey Poults via Sodium Malabsorption
Authors: Nighot Prashant K. , Moeser Adam , Ali Rizwana , Blikslager Anthony , Koci Matthew
Published: June 2021

We studied a model of turkey astrovirus type 2 (TAstV‐2)‐induced diarrhea in poults. Four days post‐infection, when infected poults exhibited diarrhea, either intact or stripped intestinal mucosa was mounted on Ussing chambers to study ion transport. Isotopic flux studies demonstrated impaired Na+ absorption with reduced short circuit current and increased conductance in astrovirus‐infected jejunum. In electron microscopic examinations, the terminal web region displayed frequent dense aggregations in TAstV‐2 infected intestine. Rearrangement of apical F‐actin was evident on immunofluorescence as thick, dense aggregations in TAstV‐2‐infected intestine. Although the total protein expression for the major apical membrane Sodium Hydrogen Exchanger, NHE3, was not changed in western analyses of astrovirus‐infected jejunum, a significant shift was observed for expression of NHE3 from detergent insoluble fractions to detergent soluble fractions of TAstV‐2 infected jejunum. Alternatively, total expression of NHE2 was up regulated in infected jejunum. Taken together, astrovirus TAstV‐2 induces malabsorptive diarrhea associated with actin re‐arrangement and redistribution of apical membrane NHE3. Further studies are currently being conducted to identify the electrogenic component of Na+ malabsorption.
NOS stimulates COX‐1‐mediated barrier function and inhibits COX‐2 in Cryptosporidium infection
Authors: Gookin Jody , Stauffer Stephen
Published: June 2021

Cryptosporidium parvum (CP) is a parasite of intestinal epithelium. CP increases intestinal mucosal synthesis of prostaglandins (PG), the majority of which is dependent on nitric oxide synthase (NOS) activity. While PG stimulate Cl‐ secretion and diarrhea in CP infection they also promote epithelial barrier function. We sought to determine the identity and location of COX expression in CP infection and whether barrier function could be ascribed to a single isoform of COX.

Expression of COX‐1 and ‐2 were quantified by real time RT‐PCR and immunoblots performed on ileal epithelium and lamina propria from control and CP‐infected piglets. PG synthesis and barrier function were measured in Ussing chambers in the presence of COX‐selective and NOS inhibitors. The role of iNOS in control of COX‐1 and ‐2 expression was investigated by examining the effect of iNOS inhibition on COX expression.

CP infection resulted in preservation of COX‐1 and inhibition of COX‐2 expression. While both COX‐1 and COX‐2 contributed to PG synthesis, NOS‐dependent epithelial barrier function was mediated by COX‐1. In contrast, iNOS inhibited epithelial COX‐2 expression in CP infection. We have shown that iNOS‐derived NO mediates the increase in PG synthesis in CP infection and NO donors can exacerbate PG synthesis and diarrhea. By downregulating expression of COX‐2, iNOS may restrain PG synthesis and diarrhea in CP infection.
Role of Panaxoside on regulating intestinal bacteria balance of hydroperitonia mice and alleviating side effect of chemotherapy
Authors: Yang Jingyun , Wu Qingtian , Hou Xia , Ma Shuxia , Wang Molin , Kang Yu‐Ming
Published: June 2021

Usually, the intestinal bacteria balance of hydroperitonia mice was modulated by panaxoside (PA) to alleviate the chemo‐treatment side effect. Hepatocarcinoma hydroperitonia model was induced by injecting H22 cells intraperitoneally. The mice were either exposed with PA before model induced or with PA and CDDP after model induced. The results showed that the rates of successful implantation of H22 cells in PA group decreased significantly compared to the control group. The quantity of the intestinal predominant ballicus and Enterococcus in the PA+CDDP group (PC) were improved, while the quantity of the Enterobacter decreased significantly. Ascitic liquid was observed on the 6th day in CDDP group, but on the 9th day in PC group. The survival time of CDDP and PC group is 16±2.1days and 38.4±2.31days respectively. The rate of bacterial translocation in liver was decreased significantly in the PC group. The splenomegaly was more obvious in PC group than in CDDP group. The spleen index also increased significantly in PC group (1.43±0.16). The quantity of lymphocytes, macrophages and platelet increased in the PC group. So panaxoside could not only regulate intestinal microecology balance, improve immune function, but also prevent the formation of hepatocarcinoma hydroperitonia, relieve the side effects of chemotherapy, reduce the incidence of cancer.
Effect of uncontrollable stress on the development of endometriosis and expression of S‐100 and COX‐2 in an animal model of endometriosis
Authors: Cuevas Marielly , Santiago Olga I , Hernandez Jesus , Rivera Liliana , Flores Ihdaliz , Thompson Kenira , Appleyard Caroline B
Published: June 2021

The contribution of stress to the symptoms and progression of endometriosis, a chronic disease characterized by pain, peritoneal inflammation, adhesions and cysts, is unclear.

AIM

Examine the effect of stress on the expression of S‐100 (a marker for myelinated nerves) and COX‐2 (an enzyme catalyzing prostaglandin biosynthesis) in an animal model of endometriosis.

METHODS

Endometriosis was induced in female Sprague‐Dawley rats by suturing uterine horn implants next to the intestinal mesentery. Two weeks later one group (endo‐stress) was subjected to uncontrollable swim stress for 10 consecutive days. Endo‐control received no stress. Sham‐stress had sutures only then stress. Fecal pellets were counted during stress as a measure of anxiety. On day 60 all rats were examined for the presence of endometriotic vesicles, and the colons assessed for damage. Immunohistochemical analysis for S‐100 and COX‐2 expression was performed.

RESULTS

Exposure to stress increased the average vesicle length and their severity. No vesicles were found in sham‐stress. Stress increased colonic damage, motility, and numbers of mast cells (p<0.05). Increased positive staining for S‐100 and COX‐2 was found in colon and uterus in endo‐stress compared to endo‐control or sham‐stress.

CONCLUSIONS

Stress may exacerbate the development of vesicles in an animal model of endometriosis through mechanisms involving cell recruitment (eg. mast cells), and release of mediators such as prostaglandins.
Use of Precision‐Cut Liver Slices To Evaluate The Protective Effects Of Betaine In LPS‐Induced Liver Injury
Authors: Kharbanda Kusum , Cannella John J , Ward Brian W , Todero Sandra L , Tuma Dean J.
Published: June 2021

LPS plays an important role in the pathogenesis of alcoholic liver injury via its effect on Kupffer cell‐mediated inflammatory responses. Consequently, released cytokines can activate specific intracellular pathways in hepatocytes that influence cell fate in different manners, e.g., proapoptotic signals via the caspase cascade, but also survival pathways. We have begun to utilize the precision cut liver slice model as a tool to dissect these interactions as well as to evaluate the potential protective effects of therapeutic agents.

Here, chow‐fed rat liver slices were prepared in accordance with the manufacturer's instructions (Vitron Inc, Tucson, AZ) and exposed to LPS in the presence and absence of 5mM betaine. Slices were harvested after 24h for hepatocellular caspase‐3 activity measurements. Media was analyzed for LDH leakage and cytokine release (TNF and IL6).

The slices exposed to media alone exhibited low caspase‐3 activity and retained viability as judged by minimal leakage of LDH. Following exposure to LPS, an induction of caspase‐3 and an increase in cytokine release (p<0.05) was observed. Betaine treatment significantly attenuated LPS‐induced caspase‐3 activation (p<0.05), but had no effect on cytokine levels. This data suggests that the protective effects of betaine on LPS are down‐stream of TNF and IL6 and may occur via upregulation of survival signals in hepatocytes.