bluuebunny/crossref_metadata_2025 huggingface dataset:
Aging increases resting oxygen consumption in type‐II skeletal muscle
Authors: Villarin Jason J , Conley Kevin E , Kushmerick Martin J , Marcinek David J
Published: June 2021

Aging affects skeletal muscle metabolic function. Other studies have shown greater generation of reactive oxygen species and the resulting oxidative damage in Type‐II muscle fibers. We tested whether the greater oxidative stress results in a greater effect of age on metabolism in type II muscle. We compared oxidative metabolism in soleus muscle (SOL, primarily type I fibers) and extensor digitorum longus muscle (EDL, primarily type II fibers) of the C57BL/6 mouse in two age groups, 7‐9 months old (adult) and 27‐29 months old (aged). Saponin‐permeabilized muscles were measured with high‐resolution respirometry under ADP‐limiting (state 4) and activated (state 3) conditions. We found no difference in resting oxygen consumption (state 4) between SOL and EDL in adult muscle when normalized to wet weight. Aged muscle showed an elevation in resting oxygen consumption in EDL (62%, p<0.05), but not in soleus. Stimulated state‐3 respiration was higher in SOL, however there was no effect of age. These results suggest there is increased proton leak in the mitochondria during state‐4 metabolism in the predominantly type‐II EDL muscle with aging.

Supported by NIH F32‐AG029052, RO1‐AG028455.
Reduced Wheel Running Activity in β1/β2 Adrenergic Receptor Knockout Mice is not Caused by Anxiety
Authors: Eisner Christoph Benedikt , Muller Florian , Huang Yuning , Briggs Josephine , Schnermann Jurgen
Published: June 2021

Mice lacking β1 and β2 adrenergic receptors (β1/β2ADR ‐/‐) exhibit reduced locomotor activity as measured by telemetry transmitter. In the present experiments we have examined whether the reduced locomotor activity resulted of augmented anxiety levels. Wild type (WT) and β1/β2ADR ‐/‐ mice were housed in running wheel (RW) cages for a 2 week period at a 12h light:12h dark (LD) cycle followed by a 2 week period at 12h dark: 12h dark (DD) conditions. The 24 hour average of RW revolutions/min was significantly lower in β1/β2ADR‐/‐ mice (LD: 3 ¡± 1; DD: 4 ± 1; n=7) compared to WT mice (LD: 18 ± 3; DD: 17 ± 4; n=8; p < 0.05). The activity showed a circadian pattern in both genotypes, but the periodic strength was significantly greater in WT (LD: 1117 ± 152; DD: 842 ± 135; n=8) than in β1/β2ADR‐/‐ (LD: 561 ± 114; DD: 414 ± 105; n=7; p < 0.05). To study anxiety levels behavior was analyzed in an open field test by videotracking for 5 min. 30‐35 min after placement into the open field, total distance traveled was 1382 ± 283 cm in WT (n=4) and 823 ± 150 cm (n=4; p<0.05) consistent with reduced locomotor activity. However, β1/β2ADR‐/‐ mice were found to spend 73 ± 8 % of total time in the open inner field while WT mice only spent 25 ± 9 % in the open field preferring proximity of walls for 75% of the time (n=4; p<0.05). In conclusion, absence of β1/β2ADR causes reduced voluntary locomotor activity and strength of circadian rhythmicity despite reduced levels of anxiety.
Short‐term aerobic exercise training decreases Glucagon‐like Peptide‐1 in obese individuals
Authors: Baynard Tracy , Carhart Robert L , Kanaley Jill A
Published: June 2021

Glucagon‐like peptide 1 (GLP‐1) is a relatively new incretin hormone secreted by the gut and is associated with appetite regulation and satiety. It is actively under study as a therapy for obesity and diabetes, yet the effects of exercise on this hormone are unknown.

Objective

To determine if a short‐term training period would influence GLP‐1 in obese individuals.

Methods

Sedentary, obese individuals (n=17; 51 yrs; body mass index 33.8 kg/m2) underwent 10 continuous d of exercise training. Training consisted of treadmill walking for 1 h/d at 75% of peak aerobic capacity. GLP‐1, glucose tolerance (2 hr meal test; 2 cans of Ensure(tm)) and aerobic capacity were assessed pre and post‐training.

Results

Body weight did not change with training, but peak aerobic capacity improved (24.1 ± 1.7 vs. 26.0 ± 1.8 mL/kg/min; p<0.05). Fasting insulin concentrations decreased (41.6 ± 5.3 vs. 31.0 ± 2.9 pmol/L; p<0.05), while glucose tolerance was unchanged (glucose AUC). GLP‐1 decreased by 27% following training (3.01 ± 0.32 vs. 2.20 ± 0.22 pmol/L; p<0.05). The change in fasting insulin was correlated to the change in GLP‐1 (r = 0.97; p<0.001).

Conclusions

Aerobic exercise training appears to elicit early changes in the gut hormone, GLP‐1, in obese persons. This may be an important mechanism by which exercise improves glucose tolerance and insulin sensitivity independent of changes in skeletal muscle. Research support: NIH R21 DK063179 (JAK); Sigma Delta Epsilon Fellowship; American College of Sports Science Mid‐Atlantic Chapter.
Exercise alters susceptibility to LPS‐induced lung injury
Authors: Pastva Amy , Griffin Michael , Giamberardino Charles , Wright Jo Rae
Published: June 2021

Lipopolysaccharide (LPS) is an airborne microbiological contaminant that has been associated with adverse respiratory symptoms and asthma exacerbations. Because exercise modulates the immune system, we hypothesized that an active lifestyle attenuates susceptibility to LPS‐induced lung injury. Mice (C57BL/6; 8 wks) were exercised at moderate intensity 5 days/wk for 4 wks for 45 min or were exposed to similar environmental conditions but remained sedentary. After training, mice were challenged with either filtered air (FA) or aerosolized LPS (~4 μg/m3) for 2.5 hrs and measurements were taken 4 and 24 hrs after aerosol exposure. Although LPS treated mice had increased cell counts, neutrophilia, protein levels, and cytokine KC levels in the bronchoalveolar lavage fluid compartment compared to FA mice, similar levels were noted between LPS treated sedentary (LS) and exercised mice (LE). However, in the lung tissue compartment histological analysis revealed that LE mice had decreased perivacular and peribronchial cellular infiltrates compared to LS mice. In preliminary lung function tests, LE mice exhibited reduced airway hyperresponsiveness to aerosolized methacholine challenge compared to LS mice. Taken together, these data suggest that exercise alters the susceptibility of mice to LPS‐induced lung injury.
Role of Gαi/s/AC/cAMP signaling in the rostral ventrolateral medulla (RVLM) in normotension and hypertension
Authors: Goodchild Ann K , Hildreth Cara M , Tallapragada VJ , Hassan Sarah F
Published: June 2021

A multitude of G‐protein coupled receptors act in the RVLM. GPCRs signal via Gα proteins mainly through three major signaling cascades. We sought to determine the role Gαi/s/AC/cAMP signaling cascades play in the RVLM in the tonic and reflex regulation of arterial pressure (AP). In urethane anaesthetized, paralysed and artificially ventilated rats, activators and inhibitors of PKA (Sp‐,Rp‐cAMPs) were injected into the RVLM. Activation evoked profound increases in AP (55±13mmHg, 5nmol, n=4) and sympathetic nerve activity (115±19%) and facilitated baroreceptor and somatosympathetic reflex function. In conscious telemetered SHR (n=9) and WKY (n=10) we compared the autonomic responses (MAP, HR and temperature) evoked by 8‐OHDPAT (0.1, 1, 3mg/kg iv) or oxotremorine (0.2mg/kg iv). Significantly enhanced dose related responses (AP and/or HR) were evoked in SHR suggesting that the GPCRs or downstream proteins are altered. In SHR, blockade of Gαi/o proteins in the RVLM, using pertussis toxin (PTX), abolished the cardiovascular effects of 8‐OHDPAT and oxotremorine at day 6 but not at day 2 following PTX administration but had no effect on the resting level of AP. These data indicate that in the RVLM, activation of 1) Gs proteins evoke profound sympathoexcitatory effects and enhanced sympathetic reflex function and 2) Gi proteins evoke enhanced responses in SHR versus WKY but may play little role in setting the level of AP.
Presynaptic inhibition of GABA release by activation of cannabinoid 1 receptors in the NTS
Authors: Chen Chao‐Yin , Dean C , Hopp Francis A , Seagard Jeanne L
Published: June 2021

In previous studies, we have shown that endocannabinoid activation of cannabinoid 1 receptors (CB1Rs) in the NTS prolongs baroreflex‐evoked sympathoinhibition through modulation of GABAergic transmission in Sprague Dawley rats. Activation of CB1Rs in studies by others in many regions of the CNS has been shown to presynaptically inhibit GABA release, disinhibiting postsynaptic neurons. Here we test the hypothesis that presynaptic inhibition of GABA inputs to 2nd‐order baroreceptor neurons contributes to the CB1Rs induced prolonging of sympathoinhibition. We recorded miniature GABAergic inhibitory postsynaptic currents (GABA mIPSCs) in anatomically‐identified 2nd‐order baroreceptor neurons in brainstem slices in the presence of ionotropic glutamate receptor blockers (NBQX, 10 μM; AP5, 50 μM) and TTX (1 μM). Data showed a concentration‐dependent decrease (p<0.05) in mIPSC frequency to increasing levels of a CB1R agonist, WIN55212‐2 (WIN2, 0.3‐30 μM) while having no consistent effect on the mIPSC amplitude. At the highest concentrations, WIN2 decreased the mIPSCs by 30% with an EC50 of 2.3 μM. The data suggest that endocannabinoids may contribute to the prolonged baroreflex‐evoked sympathoinhibition by acting on presynaptic CB1Rs to inhibit GABAergic transmission. (Support: VA Medical Research Funds).
Gonadectomy enhances cocaine‐induced locomotor activity in male rats
Authors: Menendez‐Delmestre Raissa , Segarra Annabell
Published: June 2021

Sex differences in cocaine addiction have been demonstrated in clinical as well as animal studies. However, studies on the role of testosterone in mediating these sex differences are inconclusive. This study investigated the effect of gonadectomy on cocaine‐induced hyperactivity. Adult male rats (300‐320g) were gonadectomized (GDX) or left gonadally intact (INT). Six days later, animals were placed in automated activity cages and their basal locomotor activity (LMA) was recorded. The next day, rats received a saline or cocaine (10, 15 or 30 mg/kg) injection and LMA was recorded.

Cocaine increased LMA in both groups. However, this increase was higher in GDX than in INT rats, particularly at doses of 15 and 30 mg/kg. Also, while INT rats show a progressive increase in LMA to the same dose of cocaine (sensitization) at all doses tested, only a dose of 10 mg/kg was effective in inducing behavioral sensitization to cocaine in GDX males. These results show that in the male rat, gonadal hormones participate in mediating neuroadaptations to repeated cocaine administration.

Support contributed by: NIH grants: U54NS39405 (SNRP); SO6‐GM08224 (MBRS/SCORE); and R25GM61838 (MBRS/RISE)]
Lesions of catecholamine neurons in the NTS lead to cardiac damage and sudden death in rats
Authors: Talman William T. , Zhang Wei , Nitschke Dragon Deidre , Lin Li‐Hsien
Published: June 2021

We have shown that selective lesions of nucleus tractus solitarii (NTS) neurons that express NK1 receptors lead to loss of baroreflexes, labile arterial pressure, myocardial lesions and sudden death. Because selective destruction of NTS catecholaminergic neurons expressing tyrosine hydroxylase (TH) also causes lability of arterial pressure and loss of baroreflexes, we sought to test the hypothesis that cardiac lesions and lability are not dependent on specific interruption of NK1 receptor transmission. To selectively kill TH neurons in NTS we microinjected anti‐dopamine β hydroxylase (21ng/200nl) conjugated to saporin (anti‐DBH‐SAP) into NTS. Bilateral injections in 5 rats led to lability of arterial pressure (standard deviation of mean arterial pressure of 14.6 ± 4.1 mmHg), and one rat died suddenly 9 days after injection. All treated animals demonstrated microscopic myocardial necrosis as we have reported with lesions of NTS neurons with NK1 receptors. After injection of the toxin unilaterally in NTS immunofluorescent staining confirmed that anti‐DBH‐SAP injected into the NTS decreased neurons and fibers that contain TH and DBH in the injected side of the NTS. These findings contrast to the effect of SSP‐SAP on TH in NTS and provide an excellent means to compare and contrast effects of specific cellular loss on cardiac function. This work was supported by NIH HL59593, NIH HL088090 and VA Merit Review.
A distinct subcellular pool and an α1G T‐type Ca2+ channel regulated surface expression of P‐selectin in pulmonary capillary endothelium
Authors: Zhou Chun , Chen Hairu , King Judy A. , Sellak Hassan , Kuebler Wolfgang M. , Yin Jun , Shin Hee‐Sup , Townsley Mary I. , Wu Songwei
Published: June 2021

Regulated P‐selectin surface expression provides a rapid measure of endothelial transition to a pro‐inflammatory phenotype. In general, P‐selectin surface expression results from Weibel‐Palade body (WPB) exocytosis. Yet, it is unclear whether pulmonary capillary endothelium possesses WPBs or regulated P‐selectin surface expression. We used immunohistochemistry, immunofluorescence, ultrastructural assessment, and an isolated perfused lung model to demonstrate that capillary endothelium lacks WPBs, but possesses P‐selectin. Thrombin stimulated P‐selectin surface expression in both extra‐alveolar vessel and capillary endothelium. Only in capillaries was the thrombin‐stimulated P‐selectin surface expression markedly reduced by pharmacological inhibition of T‐type Ca2+ channels, or by genetically eliminating T‐type channel α1G‐subunits. T‐type channel‐mediated cytosolic Ca2+ transients were detected by real‐time imaging of fura‐2‐loaded capillary endothelium following plasma membrane depolarization via high K+ perfusion. Depolarization also induced P‐selectin surface expression in capillary endothelium, which was abolished by T‐type channel blockade or eliminating α1G‐subunits. Collectively, these findings indicate that, in capillary endothelium, subcellular P‐selectin is WPB‐independent, and its regulated surface expression is driven by mechanism(s) distinct from WPB exocytosis; namely, Ca2+ entry through the α1G T‐type channel controls the regulated P‐selectin surface expression in capillary endothelium.
The CYP4A‐20‐HETE System in Regulation of Endothelial Progenitor Cell Functions
Authors: guo austin meng , Janic Branislave , Arbab Ali S , Edwards Paul A , Scicli Alfonso Guillermo
Published: June 2021

Endothelial progenitor cells (EPC) are essential in the repair of impaired vascular perfusion because they contribute to neovascularization and wound healing. Cytochrome P450 4As are ω‐hydroxylases that metabolize arachidonic acid (AA) to 20‐hydroxyeicosatetraenoic acid (20‐HETE). We have reported that 20‐HETE is angiogenic and that inhibitors of 20‐HETE synthesis inhibit angiogenesis. In this study, we hypothesize that the CYP4A‐20‐HETE system regulates EPC functions. We found that the mRNA coding for CYP4A11, the main human 20‐HETE synthase is present in EPC isolated from human umbilical cords. These EPC contain large amounts of immunoreactive CYP4A11 and they form and secrete 20‐HETE when incubated with AA. Further, 20‐HETE increases EPC proliferation and migration, suggesting that EPC are a target for 20‐HETE. When matrigel plugs containing 20‐HETE are injected sc in nude mice, a marked angiogenic response is observed. Injection of EPC iv results in the accumulation of these EPC to the site of angiogenic response in the plug, suggesting that 20‐HETE may induce EPC homing. Additionally, 20‐HETE induces tube formation by human endothelial cell, and EPC appear to incorporate into the tube wall, suggesting that 20‐HETE facilitate EPC involvement in vessel formation. Thus the CYP4A‐20‐HETE system may be involved in regulation of EPC functions, a novel finding with potentially high clinical relevance.
Antioxidants quercetin and dihydroquercetin inhibit ex vivo hemolysis but not plasma lipid peroxidation
Authors: Chen Yifan
Published: June 2021

Although both are antioxidants, the flavonoids quercetin (Q) and dihydroquercetin (DHQ) can produce different biological effects. For example, Q is known to inhibit heat stress‐induced heat shock proteins, whereas DHQ has no such effect. Thus, we tested if Q and DHQ differ in their protective capacities against oxidation in human blood erythrocytes and plasma. DHQ showed a greater potency of inhibiting xanthine‐oxidase‐dependent superoxide generation compared to Q (EC50: 17.4 ± 3.6 μM vs 70.8 ± 19.3 μM, P < 0.001). Hemolysis and plasma lipid oxidation were initiated with AAPH and measured in real‐time by spectrophotometry. Following 30‐min incubation, DHQ at lower concentrations and Q at higher concentrations reduced hemolysis rates. Neither Q nor DHQ inhibited the plasma lipid oxidation. Incubation of plasma with AAPH for 4 hrs caused an increase in TBARS (10.0 ± 0.7 at 4 hr vs 3.5 ± 0.4 μM at 0 min, P < 0.001), which could not be prevented in the presence of Q or DHQ. The total plasma antioxidant was equivalent to 207 ± 93 mU/ml of superoxide dismutase (EC50: 0.72 ± 0.36 mU/ml). We conclude that DHQ and Q at physiological concentrations require cellular interaction to provide protection against lipid oxidation though DHQ is a more potent antioxidant. Healthy adults have a very substantial plasma antioxidant capacity that may not be immediately improved upon taking dietary Q or DHQ. (Supported by USUHS intramural grant RO91EH)
Cardiovascular and Renal Effects Of Leptin In Females: Does Menopause Make A Difference?
Authors: Alarcon Fortepiani M Lourdes , Hinojosa‐Laborde Carmen
Published: June 2021

Menopause has been associated with increased cardiovascular (CV) and renal risk, elevated leptin levels and obesity; however the importance of leptin in postmenopausal hypertension and renal disease has not been characterized. This study tested the hypothesis that the CV and renal effects of leptin in aged female rats are enhanced after ovary removal. Old female rats implanted with telemetry devices were divided into ovariectomized (ovx) or sham operated. After 5‐day control period, rats were centrally infused with leptin (0.5ug/kg/hr) or vehicle for 5 days and BP and heart rate (HR) were continuously monitored. Urine samples were collected before and after the treatment to address renal function. Ovx increased food intake, adiposity index and decreased activity, however, BP and HR were similar in ovx and sham rats. Leptin decreased food intake in ovx (44%) and sham (28%). Despite weight loss, leptin induced similar increases on HR in both groups (approximately 10 bpm each) while elevating BP in ovx (4.7 ± 0.8 mmHg) but not in sham rats (‐0.8 ± 0.9 mmHg). Surprisingly, leptin decreased urinary protein excretion in ovx (48.6 ± 2 vs 75.6 ± 2 ug/24h) and sham (46.7 ± 5 vs 87.8 ± 5 ug/24h) vs vehicle. These results suggest that central leptin activation in aged rats contributes to the development of postmenopausal hypertension but may protect from age related renal dysfunction. This work was supported by NIH AG029250‐01 award.
Reduction of GRP78 increases expression of ATF4, GRP94, GADD153 in mIMCD3 cells.
Authors: Tse Jonathan , Cai Qi , Weber Craig , Lynch Ronald M. , Brooks Heddwen L.
Published: June 2021

We previously demonstrated that mRNA and protein expression of the 78kDa glucose‐regulated protein (GRP78) increased in the renal IM of 7 day dDAVP infused AQP1 null mice. GRP78 is an ER chaperone protein with an important role in protein folding, Ca2+ binding, and ER homeostasis. In order to investigate the role of GRP78 in osmotic tolerance, GRP78 siRNA was transfected into mIMCD3 cells to knockdown GRP78 expression. mIMCD3 cells were harvested 24hrs and 48hrs after transfection for mRNA and protein analysis respectively. We found, however, that siRNA knockdown of GRP78 resulted in increased expression of several genes involved in UPR signaling pathways. Quantitative real time PCR showed an increase in the expression of 94kDa glucose‐regulated protein (GRP94), activating transcription factor 4 (ATF4), and growth arrest and DNA damaging protein (GADD153); 3.36 ± 0.03, 1.43 ± 0.1, and 3.51 ± 0.03 fold change over control, respectively, p<0.05. Immunoblotting confirmed an increase in protein expression of GRP94 and GADD153 after GRP78 knockdown. Preliminary trypan blue staining data suggests that despite the observed GADD153 upregulation, no significant cell death occurs 48 hours after GRP78 siRNA transfection. To investigate the possible role of Ca2+ in the activation of UPR pathways in mIMCD3 cells Fura‐PE3AM will be used to monitor intracellular Ca+2 flux in GRP78 siRNA transfected mIMCD3 cells.
Urinary concentration is impared in mice lacking adenylyl cyclase 6
Authors: Rieg Timo , Tang Tong , Murray Fiona , Insel Paul A , Hammond H Kirk , Vallon Volker
Published: June 2021

The adenylyl cyclase isoform 6 (AC6) is expressed in all renal tubular segments and the collecting duct (CD). To define the in vivo role of this enzyme in arginine‐vasopressin (AVP)‐induced urine concentration, we studied AC6 wild‐type (WT, n=8) and knockout (AC6−/−, n=8) mice. With free access to food and water AC6−/−, compared to WT, have higher fluid intake (13.5±0.5 vs. 5.0±0.3 ml/day, P<0.01), lower urine osmolality (Uosm: 875±94 vs. 2027±160 mmol/kg, P<0.01) but similar GFR (10.0±0.6 vs. 11.4±1.1 μl/min/g bw) and plasma osmolality (Posm: 306±1 vs. 305±2 mmol/kg), indicating intact fluid balance. 24 h water restriction revealed a lower Uosm in AC6−/− vs. WT (2577±110 vs. 3095±98 mmol/kg, P<0.01) in spite of a greater urinary AVP/creatinine ratio (1120±24 vs. 405±71 nmol/mmol, P<0.05), associated with greater body weight loss (‐4.3±0.5 vs. ‐2.8±0.1 g, P<0.05) and greater Posm (339±3 vs. 323±2 mmol/kg, P<0.01). Oral water‐loading (3% of bw; to suppress endogenous AVP) lowered urine osmolality to similar levels in AC6−/− vs. WT (259±38 vs. 317±63 mmol/kg) but total urinary cAMP excretion and the increase in Uosm with co‐administration of the agonist dDAVP (0.1 μg/kg i.p.) were lower in AC6−/− (1.0±0.1 vs. 2.7±0.7 pmol/min/g bw, P<0.05; 2020±61 vs. 3127±94 mmol/kg, P<0.01). These results demonstrate that expression of AC6 is important for urinary concentration in vivo, including in response to water restriction and AVP administration. (Supported by grants from NIH, VA San Diego Healthcare System and National Kidney Foundation)
Structure and function of inner medullary intercluster and intracluster vasa recta
Authors: Pannabecker Thomas L. , Dantzler William H.
Published: June 2021

Inner medullary (IM) UTB‐positive descending vasa recta (DVR) lie predominantly in intercluster regions of the outer zone (OZ) whereas fenestrated ascending vasa recta (AVR) and capillaries lie in both the intracluster and intercluster regions of the OZ. In the inner zone (IZ) nearly all vessels are fenestrated. In the OZ, fenestrated AVR participate in countercurrent exchange (CCE) with UTB‐positive DVR solely in intercluster regions. Rollhauser et al 1964 reported DVR branching at various levels to form capillary networks all the way down to the papilla tip; however, we find UT‐B‐positive DVR exhibit minimal branching ‐ two branches occur at most along their entire length. Some proportion of descending blood appears carried by UTB‐negative, PV1‐positive vessels throughout the IM; CCE between these and ascending fenestrated vessels possibly occurs throughout the IM at least within the intercluster region. Capillaries join AVR at various levels all the way up to the OM‐IM boundary. Relatively unbranched AVR lie in intercluster regions and are largely absent in the intracluster regions of the OZ which are populated with branching AVR and capillaries. Absence of CCE (between vessels) in the intracluster regions and existence of CCE in the intercluster regions argues that AVR in these 2 regions are functionally distinct. The pathway by which excess water leaves the IM and passes into the OM likely resides in the intracluster region. DK16294
Epac Regulation of Urea Transport and the UT‐A1 Urea Transporter in Rat Inner Medullary Collecting Duct.
Authors: Wang Yanhua , Klein Janet D. , Martin Christopher F. , Kent Kimilia J. , Wall Susan M. , Sands Jeff M.
Published: June 2021

Urea is critical to the urinary concentrating mechanism and to the regulation of water balance. Vasopressin, through cAMP, stimulates urea transport across perfused rat terminal inner medullary collecting ducts (IMCD) by increasing UT‐A1 phosphorylation and membrane accumulation. Cyclic AMP is known to act through protein kinase A (PKA); however, it can also activate Epac (exchange protein activated by cAMP). We tested whether Epac is involved in regulation of urea transport and UT‐A1 transporter activity in rat IMCD. Functional analysis showed that the Epac activator, Sp‐8‐pCPT‐2 O‐Me‐cAMPS, significantly increased urea permeability in isolated, perfused rat terminal IMCDs by 29%. Incubation of rat IMCD suspensions with the Epac activator significantly increased UT‐A1 in the plasma membrane (biotinylated UT‐A1) and UT‐A1 phosphorylation. To further explore the signaling pathway, we tested whether forskolin could stimulate ERK 1/2 phosphorylation. Forskolin increased ERK 1/2 phosphorylation 25%; this increase was not inhibited by a PKA inhibitor, H‐89, indicating that ERK 1/2 phosphorylation was through Epac. The MEK 1/2 inhibitor, U0126, inhibited forskolin‐stimulated UT‐A1 phosphorylation. We conclude that activation of Epac increases urea transport, UT‐A1 phosphorylation, and UT‐A1 plasma membrane accumulation. The increase in UT‐A1 phosphorylation is mediated by the MEK‐ERK pathway.
Can high carbohydrate diets affect Ca and Pi serum levels by controlling their intestinal absorption in end stage renal disease?
Authors: Douard Veronique , Sugiura Shozo H , Asgerally Abbas Navroze , casirola Donatella , Ferraris Ronaldo P
Published: June 2021

The product of [Ca] x [Pi] levels in the blood is a major risk factor for cardiovascular calcification and is a function of serum Pi concentration. Hyperphosphatemia is common in end stage renal disease (ESRD), and must be reduced to lower mortality risk. Since fructose (F) inhibits intestinal Pi transporter NaPi2b expression in neonates and F is a major constituent of American diet, we studied intestinal Pi and Ca absorption in 5/6 nephrectomized adult rats and determine if dietary F modulates serum [Ca] x [Pi]. Six wk after nephrectomy, rats displayed ESRD symptoms, then isocaloric and isonitrogenous glucose (G) and F diets were fed for one month to ESRD and sham‐operated rats. Blood urea nitrogen (BUN) increased in ESRD rats within 2 wk after surgery and was affected by F: ESRD (56 mg/dL vs 40) and sham (28 vs 17). ESRD rats had higher serum Pi levels, but F failed to reduce serum Pi. Kidney mass index was 35% greater in ESRD fed G, and 75% greater in ESRD fed F. NaPi2b mRNA and protein abundance as well active and passive Pi transport were independent of diet and surgery. Ca serum levels were not affected by nephrectomy or diet. However, while duodenal transport of Ca was independent of surgery, this transport and calbindin 9K expression decreased with dietary F. Hence, dietary F exacerbates the clinical symptoms of ESRD (Pi and BUN) and alters intestinal Ca absorption, perhaps by reductions in calbindin expression (NIH RDK075617).
Hepatocyte growth factor (HGF) suppresses hepcidin expression in hepatocytes by interfering with BMP‐mediated hepcidin induction
Authors: Goodnough Julia , Nemeth Elizabeta , Ganz Tomas
Published: June 2021

Hepcidin, a 25‐amino‐acid peptide synthesized in the liver, is the iron regulatory hormone. Suppression of hepcidin in the setting of anemia appears to depend on a soluble factor expressed in the bone marrow during erythropoiesis. Here we describe that hepatocyte growth factor (HGF) suppresses hepcidin mRNA in a dose‐response fashion, in the concentration range detected in human serum. A similar response is observed in primary cells or cell lines transfected with a hepcidin promoter‐luciferase reporter. HGF is a ligand for the receptor tyrosine kinase (RTK), Met. In HepG2 cells transfected with the hepcidin‐luciferase reporter, the luminescence is specifically suppressed by HGF and not other ligands of RTKs, PDGF and EGF. BMPs 2, 4, 6 and 9 are strong inducers of hepcidin, and the BMP2/4 pathway also mediates hepcidin induction by iron‐transferrin. We report that HGF prevents a BMP2‐mediated increase in hepcidin mRNA and the activity of the luciferase reporter. HGF also prevented the induction by BMP2 of an unrelated BMP‐sensitive luciferase reporter. In primary hepatocytes, the BMP‐sensitive transcript ID1 is also downregulated by HGF. Thus, HGF may be a novel suppressor of hepcidin, modulating hepcidin expression by interfering with BMP signaling. We speculate that HGF could mediate the suppression of hepcidin in chronic liver diseases.

This research is supported by National Institutes of Health grant #1F30DK082151.
Toll‐Like Receptors 1,2,4,6 expression in Human Fibroblast of the Large Intestine
Authors: Helton‐Rieter Megann Kathleen , Reaves Titus A , LeBel David
Published: June 2021

Crohn's Disease (CD) is characterized by dysregulated intestinal inflammation with an unknown etiology. Intestinal fibrosis is a major patho‐physiological complication of CD with features that include an over production of collagen, excessive contraction of intestine tissue, and intestinal stricture. The subepithelial fibroblast is the cell type responsible for producing the increased collagen and therefore, the associated fibrosis. Despite these observations, the cause and progression of intestinal fibrosis in CD is not clearly defined. Interestingly, persistent intestinal fibrosis has been reported in Salmonella infections in mice. Given that toll‐like receptors (TLRs) likely modulate the response to this pathogen, we characterized the expression of TLRs 1, 2, 4, and 6; and a TLR‐2 associated protein termed Triggering Receptor Expressed on Myeloid Cells (TREM‐1) in human intestinal fibroblasts. Results revealed that fibroblast express TLR's 2, 4, 6, and TREM‐1, but minimal to no expression of TLR‐1. Fibroblasts exposed to interleukin‐6 ((IL‐6), inflammatory cytokine abundantly expressed in CD), revealed a pronounced reduction in expression of TLR‐6 when compared to TLRs 2, 4 and TREM‐1. Since activation and proliferation of fibroblasts is the key component of fibrogenesis, then excessive exposure to microbes may result in aberrant collagen production thereby initiating intestinal fibrosis.
Interferon‐gamma (IFN γ) induced epithelial barrier dysfunction in T84 human intestinal epithelial cells (IECs) occurs via phosphatidylinositol 3‐kinase (PI3‐K) mediated activation of adenosine monophosphate‐activated protein kinase (AMPK)
Authors: Scharl Michael , Barrett Kim E. , McCole Declan F.
Published: June 2021

IFNγ plays a pivotal role in the pathophysiology of inflammatory bowel disease (IBD) and some of the effects of IFNγ on intestinal epithelial barrier function are dependent on PI3‐K. Here we investigated the role of the cellular energy sensor, AMPK in the effects of IFNγ on epithelial barrier function.

Protein analysis was performed by Western blotting and AMPK knock‐down by siRNA. Transepithelial resistance (TER) was assessed by voltohmeter and epithelial permeability as the flux rate of 10 kDa FITC‐dextran.

IFNγ (1000 U/ml) induced activation of AMPK in a manner independent of changes in intracellular ATP (the classic trigger for AMPK activation). Inhibition of PI3‐K with LY294002 (20 µM) blocked IFNγ‐induced AMPK phosphorylation (n=9; p<0.05; 6h), and mitigated the IFNγ‐induced decrease in TER across T84 monolayers (n=9; p<0.05). Knock‐down of AMPK diminished the IFNγ‐induced increase in epithelial permeability (72 h; n=3; p<0.05) and prevented IFNγ‐induced downregulation of the tight junction proteins occludin and ZO‐1 (n=3; p<0.05).

We conclude that IFNγ sequentially activates PI3‐K and AMPK to impair tight junction protein expression and thus barrier function in IECs. Our data indicate a novel role for AMPK as a mediator of cytokine signalling, independent of changes in cellular energy status, and thus may have implications for mucosal defense in the setting of inflammation.

Supported by the CCFA and NIH.
Age‐dependent regulation of sodium‐potassium adenosinetriphosphatase and sodium‐hydrogen exchanger mRNAs in equine nonglandular mucosa
Authors: Andrews Frank Michael , Peretich Amanda Lynn , Abbott Lora L , Dhar Madhu S
Published: June 2021

Gastric ulcers, common in young race horses, occur in the nonglandular (NG) region. Histopathology in tissues exposed to hydrochloric acid (HCl) and volatile fatty acids (VFAs) in vitro showed compromised sodium transport, likely regulated by cellular Na‐K‐ATP (NAKA) and/or Na‐H exchanger (NHE) enzymes. We determined relative gene expressions of NAKA and NHE in equine NG mucosa exposed to HCl and VFAs at three different pHs (1.5, 4, or 7.0). Five (≤5 yrs of age) and five (≥12 yrs of age) mixed breed and gender horses were used. NG mucosa near the Margo plicatus was collected and exposed in Ussing chambers to normal Ringer's solution (control), solution with a mixture of VFAs and solution with 40 mM of acetic acid (treatment). NAKA and NHE mRNA expressions were determined using real time PCR.

We found increased expression of both target genes after exposure to VFAs, compared to controls. Interestingly, younger horses had significantly (P≤0.05) down‐regulated NAKA, while expression of NHE was increased. On the contrary, both NAKA and NHE were up‐regulated in older horses. Young horses may be more susceptible to gastric ulcers due to an inability to up‐regulate active sodium transport through NG mucosal cells. Novel treatments could be developed to enhance NG cell function and prevent gastric ulcers in horses. Funded by Centers of Excellence in Livestock Disease and Human Health, University of Tennessee.
A novel signaling pathway mediates the molecular effect of LPS in inhibition of eNOS activation involving cofilin and VASP
Authors: Kwok Willson , Clemens Mark G
Published: June 2021

Liver sinusoidal endothelial cells (LSECs) are the major source of endothelin‐1 (ET‐1) in the liver. ET‐1 is a potent vasoconstrictor; however, it also mediates vasodilation of the liver sinusoids when it induces endothelial nitric oxide synthase (eNOS) activation in the LSECs. Lipopolysaccharide (LPS) inhibits ET‐1‐mediated eNOS activation but the molecular mechanisms are unclear. We hypothesized that cofilin and vasodilator‐stimulated phosphoprotein (VASP) contribute to decreased eNOS activity. To test this hypothesis, we used a Rho‐kinase (ROCK‐2) inhibitor, Y27632. LPS increased ROCK‐2 activity and expression (+30%). Inhibition of ROCK‐2 not only augmented the ET‐1‐mediated eNOS activation and nitric oxide (NO) production (+63%), it also reversed the inhibitory effect of LPS (+61%). The treatment reduced cofilin phosphorylation (‐36%) and increased VASP phosphorylation (+34%), suggesting an upregulation of actin depolymerization and downregulation of actin polymerization, respectively. This was confirmed by confocal microscopy showing a higher level of G‐actin interacting with eNOS following this treatment. In conclusion, cofilin and VASP partly mediate the downstream effects of ROCK‐2 to reduce G‐actin levels following LPS treatment resulting in the inhibition of the ET‐1‐mediated eNOS activation. Supported by DK38201
Cardiovascular and metabolic responses to chronic PYY3‐36 infusion
Authors: Dubinion John Harvey , daSilva Alexandre A , doCarmo Jussara M , Hall John E
Published: June 2021

Peptide YY3‐36 (PYY3‐36) is secreted by the gastrointestinal tract in response to feeding and has been shown to have anorexic actions during acute systemic injections. The importance of PYY3‐36 in chronic regulation of food intake and cardiovascular function, however, is poorly understood. The purpose of the current study was to test the hypothesis that chronic infusion of PYY3‐36 leads to sustained reductions in food intake and elevations in blood pressure (BP) and heart rate (HR). Sprague Dawley rats (n=7) were instrumented with arterial and venous catheters for continuous monitoring of BP and HR. After recovery and 5 days of control measurements, PYY3‐36 was infused IV for 7 days at a dose of 10 nM/hr. Post‐control measurements were taken for 5 additional days. PYY3‐36 infusion caused a sustained 20% reduction in caloric intake after day 3 (82 ± 2 kcal to 65 ± 2 kcal). BP remained unchanged during PYY3‐36 infusion (+2.8 ± 0.1 mmHg, average change on days 5‐7 of infusion), whereas HR increased significantly (20 ± 1 bpm). Food intake returned to control level within one day after stopping PYY3‐36 infusion. Heart rate, however, did not return to control values until the 2nd day following the termination of PYY3‐36 infusion. Our results indicate that chronic PYY3‐36 infusion caused a sustained reduction in appetite while increasing HR but had no significant effect on chronic blood pressure regulation. (NHLBI ‐ PO1 HL51971).
Efflux of cAMP in the exocrine pancreas: Role of secretin and atrial natriuretic factor (ANF)
Authors: Lange Maria S , Rodriguez Myriam , Ventimiglia Maria , Vatta Marcelo , Davio Carlos , Bianciotti Liliana
Published: June 2021

We previously reported that ANF dose‐dependently reduces secretin secretory response and intracellular signaling in the exocrine pancreas through NPR‐C receptors coupled to the PLC/PKC pathway. In this study we evaluated if cAMP efflux contributed to ANF response in isolated pancreatic acini. Results showed that ANF favored the efflux of cAMP formed by secretin. The export of cAMP was also observed with secretin alone but not with isoproterenol or amthamine. Evoked‐cAMP efflux was blocked by probenecid, suggesting that multidrug resistant proteins (MRPs) were involved. RT‐PCR analysis revealed that MRP‐4 but not MRP‐5 (transport cyclic nucleotides) was expressed in the pancreas. Time course studies in the presence of phosphodiesterase (PDE) activity showed that tissue cAMP increased peaking at 3 min onset secretin stimulation and declining thereafter. With ANF, secretin‐induced cAMP augmented reaching a peak at 1 min after which it rapidly decreased. Extracellular cAMP accumulation occurred earlier and persisted throughout the experiment whereas with secretin alone it was delayed. Similar findings were observed in studies with PDE inhibition, except that cAMP content was higher and its appearance delayed in tissues and the media. Present findings suggest that cAMP export is likely mediated by MRP‐4 and further it may contribute to ANF‐induced desensitization of secretin response in the exocrine pancreas.

(CONICET PIP5929/ UBACYT B079)
Expression and localization of natriuretic peptide receptor types A and C in human stomach
Authors: Alli Abdel A , Carter Gay M , Gower William R
Published: June 2021

Evidence indicates that ANP regulates gastric acid secretion and motility. Little is known regarding the expression and function of ANP receptors in the human stomach. The aim of this study was to examine the expression and localization of NPRA and NPRC receptors in human stomach. NPRA and NPRC transcripts were detected in antrum and body. Immunoblot results demonstrated a 130 kDa protein corresponding to NPRA and a 66 kDa protein corresponding to NPRC in both regions. NPRA was localized to the cytoplasm and membranes of smooth muscle cells, vascular structures, and a population of mucosal epithelial cells located primarily in the lower third of the mucosal glands. By double‐immunostaining, the pyloric gland cells were identified as somatostatin‐containing D cells and the oxyntic gland cells identified as both somatostatin‐containing D cells and H,K‐ATPase‐containing parietal cells. A similar localization was demonstrated for immunoreactive NPRC, except that NPRC was not located on parietal cells. We conclude that NPRA and NPRC genes are expressed in normal human gastric antrum and body. Both receptors are expressed in gastric smooth muscle cells as well as somatostatin cells. NPRA receptors are, in addition, expressed on parietal cells. These results suggest a physiologic role for ANP in the regulation of gastric somatostatin and acid secretion as well as motility in the human stomach.
Contribution of Estrogen receptor β (ERβ) on platelet mitochondrial function and microparticle production in aged female mice
Authors: Jayachandran Muthuvel , Preston Claudia C , Hunter Larry W , Jahangir Arshad , Owen Whyte G , Korach Kenneth S , Miller Virginia M
Published: June 2021

Estrogen receptor beta (βER) is present in platelets but little is known about how βER contributes to platelet physiology. Platelet mitochondrial function and circulating platelet‐derived microparticles were analyzed in aged (24 months old) female wild type (WT) and estrogen receptor beta knockout (βERKO) mice. In intact, unstimulated platelets, ERβ did not co‐localized with mitochondria. Platelet lactate production, mitochondrial membrane potential and superoxide generation were similar in both groups, but activities of cytochrome bc1 and cytochrome c oxidase were significantly less (P<0.05) in platelets of βERKO mice. Total numbers of platelet‐derived microparticles and those positive for procoagulant phosphatidylserine (PS) were two to three times higher (P<0.05) in βERKO than WT mice. Greater than 40% of procoagulant microparticles were derived from cells other than platelets in both groups of mice. Thrombin generating capacity of microvesicles was also greater in βERKO mice. Thus, loss of βER leads to significant changes in activities of enzymes of the mitochondrial electron transport system, and to increases in circulating procoagulant microparticles which may contribute to an increased thrombotic risk with aging in persons with βER gene polymorphisms. This study is supported by HL78638, AHA30503Z and Mayo Foundation.
Oxidative stress in genetically obese SHROB rats
Authors: Theodorou Maria , Escobedo Liza Victoria , Moore Alexandra , Koletsky Matthew S , Jackson Sabrina , Koletsky Rachel E , Koletsky Richard J , Ernsberger Paul
Published: June 2021

Oxidative stress is elevated in human obesity and several animal models. Oxidative stress, through the damaging effects of oxygen radicals, may contribute to obesity‐associated conditions. These include elevated blood pressure, lipids, glucose, and insulin resistance, all of which are present in the spontaneously hypertensive obese (SHROB/Kol) rat model of prediabetes. In the present study, SHROB (N=27) were compared to lean SHR/Kol (N=11) littermates. Oxidative stress was measured by assay of peroxides in plasma and the lipid fraction of kidney and liver tissue. Plasma and organ lipid peroxides were increased up to 3‐fold in untreated SHROB rats relative to lean SHR littermates. The activity in the liver cytosol of the protective enzyme superoxide dismutase was not different (SHROB: 63±5 vs SHR: 53±7 U/mg protein). Another protective enzyme, catalase, was also unchanged in the liver cytosol (SHROB: 95±6 vs SHR: 93±13 U/mg protein). Thus, the enzymes responsible for breaking down superoxide and hydrogen peroxide fail to up‐regulate in response to increased oxidative stress in SHROB. Subsequent oxidative damage may contribute to insulin resistance in the liver and renal and hepatic dysfunction.
Impaired cardiomyocyte autophagy in diabetes is associated with increased hexosamine biosynthesis and protein O‐GlcNAc levels
Authors: Shan Dan , Dell'Italia Louis J. , Chatham John C.
Published: June 2021

We have previously shown that cardiomyocytes from type‐2 diabetic db/db mice exhibit impaired hypertrophic response, which was associated with increased protein levels of β‐N‐acetyl‐glucosamine (O‐GlcNAc). Activation of autophagy has been reported to play a critical role in the hypertrophic response of the myocardium; therefore, the purpose of this study was to test the hypothesis that diabetes leads to impaired autophagy, mediated in part via activation of O‐GlcNAc synthesis. Cardiomyocytes from non‐diabetic mice subject to glucose deprivation (GD) for 3 hrs, exhibited a significant increase in Beclin‐1 (8‐fold; p<0.05) and LC3 (2‐fold; p<0.05), consistent with activation of an autophagic response. However, cardiomyocytes from db/db mice had significantly lower levels of Beclin‐1 and LC3 following GD compared to non‐diabetic cardiomyocytes, consistent with impaired autophagy. Interestingly, increasing hexosamine biosynthesis in normal cardiomyocytes with 5mM glucosamine attenuated the GD‐induced increase in Beclin‐1 and LC3; conversely, inhibition of hexosamine biosynthesis in db/db cardiomyocytes with 20µM DON, partially restored the sensitivity to GD. Treatment of non‐diabetic cardiomyocytes with 100µM PUGNAc, an O‐GlcNAcase inhibitor, which markedly increases O‐GlcNAc levels, also attenuated the response to glucose deprivation. These studies suggest that diabetes leads to an impaired response of cardiomyocytes to GD‐induced autophagy, which may be mediated in part by increased hexosamine biosynthesis and O‐GlcNAc levels. Thus, O‐GlcNAc mediated inhibition of autophagy may contribute to the abnormal response of diabetic hearts to stress. (Supported by NIH RO1 HL067464; P50 HL077100)
Direct effects of endocannabinoid receptor antagonism on the glucose transport system in insulin‐resistant skeletal muscle of the obese Zucker rat
Authors: Lindborg Katherine Ann , Henriksen Erik J
Published: June 2021

The purpose of this study was to determine the role of the endocannabinoid receptor‐1 (CB1) in the regulation of the glucose transport system in insulin‐resistant skeletal muscle of obese Zucker rats. CB1 receptor protein expression in obese soleus was significantly lower (25%, p<0.05) compared to insulin‐sensitive lean Zucker rats. Soleus muscle from obese Zucker rats was incubated in the presence or absence of insulin (5 mU/ml) and in the presence or absence of various concentrations of the CB1 receptor antagonist SR141716. In the absence of insulin, SR141716 activated glucose transport (2‐deoxyglucose uptake) by 29% at 100 nM, with an EC50 of ~50 nM. Insulin‐stimulated glucose transport was enhanced 33% by 200 nM SR141716, with an EC50 of ~25 nM. Potential CB1 receptor antagonist targets in the classical insulin signaling pathway and insulin‐independent pathway for GLUT4 translocation were assessed. SR141716 treatment did not alter tyrosine phosphorylation of the insulin receptor or phosphorylation of Akt ser473 or glycogen synthase kinase‐3 ser9. Likewise, CB1 receptor antagonism did not affect phosphorylation of AMPKα thr172 or p38 MAPK thr180/tyr182. While the CB1 receptor is involved in modulation of the glucose transport system in insulin‐resistant skeletal muscle of the obese Zucker rat, this regulation appears to be independent of the classical pathways mediating glucose transport activities.
Expression profiling analysis of the effects of intestine‐specific Pdx1 inactivation in mouse proximal small intestine
Authors: Chen Chin , Sibley Eric
Published: June 2021

Little is known about the role of transcription factor pancreatic and duodenal homeobox 1 (Pdx1) in modulating intestinal gene expression. We aimed to identify intestinal Pdx1 target genes in viable mice with normal pancreatic development. Pdx1 is conditionally inactivated in the intestine of Pdx1flox/flox;VilCre mice by Cre recombination mediated by a mouse villin 1 gene promoter. To identify intestinal genes differentially regulated by Pdx1, microarray analysis and real‐time RT‐PCR were performed with total RNA isolated from the first 5 cm of the small intestine from adult Pdx1flox/flox;VilCre and littermate control mice. Among more than 39,000 mouse transcripts screened, expression of 20 genes decreased while that of 6 genes increased more than 2‐fold in the proximal small intestine of Pdx1flox/flox;VilCre mice, compared to that of control mice (p<0.05). Pathway analysis based on 19 differentially regulated genes with known relational interactions generated a network connecting 13 of them with Pdx1, indicating a top functional significance involving cell death/survival. Molecular functions involving transport represented the most statistically significant category by clustering 18 differentially regulated genes with known functions. The results demonstrated that Pdx1 has novel functional roles in the proximal small intestine in addition to maintaining glucose homeostasis in the pancreas.