bluuebunny/crossref_metadata_2025 huggingface dataset:
Metformin treatment of angiotensin II hypertensive rats decreased electric field stimulation mediated contraction in corpus cavernosum
Authors: Labazi Hicham , Wynne Brandi Michele , Webb R. Clinton
Published: June 2021

The renin‐angiotensin system plays an important role in cardiovascular function. However, excessive increases in angiotensin II (AngII) levels are associated with increased vasoconstriction and endothelial dysfunction with concurrent reactive oxygen species (ROS) production and decreased nitric oxide (NO) bioavailability, leading to diseases such as hypertension, atherosclerosis and erectile dysfunction. AMP‐activated protein kinase (AMPK) was shown to improve endothelial function, partly through suppression of ROS and increased NO bioavailability. We hypothesized that AMPK activation may protect against increased AngII mediated contraction in corpus covernosum. Corpus cavernosum was isolated from rats that were infused with saline or AngII (65ng/min) and treated with or without the AMPK activator metformin (500mg/kg/day) for 7 days. We observed no difference between the three groups (sham, Ang II and AngII + metformin) in phenylephrine mediated contraction. However, AngII caused an increase in electric field stimulation (EFS) mediated contraction as compared to sham (4.32±0.73 mN vs 2.70±0.52 mN) and treatment with metformin decreased this response, in the absence or presence of L‐NAME. From these data, we speculate that metformin may protect against increased AngII mediated contraction through AMPK activation and ROS suppression. Grant support: RO1HL071138
Effect of betulinic acid on high glucose‐induced vascular smooth muscle cells proliferation
Authors: Yoon Jung Joo , Lee Yun Jung , Kim Jin Sook , Kang Dae Gill , Lee Ho Sub
Published: June 2021

Betulinic acid (BA) is a pentacyclic triterpene found in Lycopus lucidus Turcz. Hyperglycemia has been shown to stimulate vascular smooth muscle cells proliferation, a paramount feature in atherosclerosis. The aim of this study was to determine the effects of BA on high glucose‐induced human aortic smooth muscle cell (HASMC) proliferation. In this study, [3H]‐thymidine incorporation under 25 mM glucose (HG) was significantly accelerated compared with 5.5 mM glucose and this increase was inhibited by BA. Invasion and migration assay also suggested an anti‐proliferative effect of BA. BA elevated the protein content of p21waf1/cip1, p27kip1, decreased cyclin D1/E and CDK2/4, suggesting that BA inhibited HASMC proliferation by regulating the cell progression from G1 to S phase. We showed that BA significantly inhibited HG‐induced matrix metalloproteinase (MMP)‐2/‐9 activity in a dose‐dependent manner. BA inhibited the HG‐induced hydrogen peroxide production and DCF‐sensitive intracellular ROS formation. Furthermore, BA suppressed the translocation and promoter transcriptional activity of NF‐κB under HG condition. BA also decreased HG‐induced phosphorylation of IκB‐α. In conclusion, the present data suggested that BA regulated cell cycle regulatory proteins, and significantly reduced HASMC proliferation, via inhibition of ROS and NF‐κB activation in hyperglycemia conditions.
Bisphenol A activates Maxi‐K channels in coronary smooth muscle
Authors: Asano Shinichi , Dick Gregory M
Published: June 2021

Bisphenol A (BPA) is used to manufacture plastics, including food containers into which it leaches. Exposure to this estrogenic endocrine disruptor is associated with diabetes and heart disease. Estrogen and estrogen receptor modulators increase the activity of large conductance Ca2+/voltage‐sensitive K+ (Maxi‐K) channels, but the effects of BPA on Maxi‐K channels are unknown. We tested the hypothesis that BPA activates Maxi‐K channels through a mechanism that depends upon the regulatory β1 subunit. Patch clamp recordings of Maxi‐K channels were made in human and canine coronary smooth muscle cells as well as in AD‐293 cells expressing pore‐forming α or α plus β1 subunits. BPA (10 μM) activated an outward current in smooth muscle cells that was inhibited by penitrem A (1 μM), a Maxi‐K blocker. BPA increased Maxi‐K activity in inside‐out patches from coronary smooth muscle, but had no effect on single channel conductance. In AD‐293 cells with Maxi‐K channels composed of α subunits alone, 10 μM BPA did not affect channel activity. When channels in AD‐293 cells contained β1 subunits, 10 μM BPA increased channel activity. Effects of BPA were rapid (<1 min) and reversible. A higher concentration of BPA (100 μM) increased Maxi‐K current independent of the β1 subunit. These data indicate BPA increases the activity of Maxi‐K channels and may represent a basis for some potential toxicological effects.
Hypotensive, hypolipidemic, and vascular protective effect of Morus alba L. in rats fed a high‐fat diet
Authors: Lee Yun Jung , Choi Deok Ho , Hwang Sun Mi , Ahn You Mee , Kim Jin Sook , Kang Dae Gill , Lee Ho Sub
Published: June 2021

Morus alba L. has been used in traditional Chinese medicine and almost all parts of this plant is useful in cardiovascular, liver and spleen disorders. Present study was designed to investigate the inhibitory effect of a water extract from Morus alba L. (WMA) on vascular dysfunction in rat models fed a high‐fat diet. Male rats were fed a high‐fat diet consisting of 60 kcal% fat, with or without 100, 200 mg/day/kg WMA of diet, for 9 weeks. Chronic treatment with low (100 mg/kg/day) or high (200 mg/day/kg) doses of WMA markedly significantly attenuated hypertension and the impairments of acetylcholine‐ and SNP‐induced relaxation of aortic rings in rats fed a high‐fat diet. WMA attenuated plasma levels of triglyceride (TG) and augmented plasma level of high‐density lipoprotein (HDL), whereas, no effects in blood glucose levels and plasma low‐density lipoprotein (LDL) levels in rats fed a high‐fat diet. Interestingly, WMA suppressed increased cell adhesion molecules such as intracellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), and E‐selectin expression in aorta. Taken together, these results suggested that Morus alba L. could improve high‐fat diet‐induced hypertension, hyperlipidemia, and vascular dysfunction through inhibition of cell adhesion molecules expression and induction of vascular relaxation.relaxation.
The effects of contractile activity on mitochondrial adaptations in PGC‐1alpha‐deficient muscle cells
Authors: Uguccioni Giulia , Hood David A.
Published: June 2021

PGC‐1α is a well established mediator of mitochondrial content and function in skeletal muscle. However, the essentiality of the coactivator for mitochondrial biogenesis (MB) in response to chronic contractile activity (CCA) is still in question. Thus, we depleted skeletal muscle C2C12 cells of PGC‐1α using siRNA, then stimulated the myotubes to induced MB. A 60% reduction in PGC‐1α using siRNA resulted in a compensatory increase in AMPK phosphorylation, but had no effect on the mRNA expression of PGC‐1β, PRC, NRF‐1/2 or YY1, proteins that may have served to compensate for low PGC‐1α levels. In response to CCA, we found that a normal induction of PGC‐1α in response to CCA was required to observe the typical changes in MB, as revealed by attenuated increases in COX activity (p<0.05) and mitochondrial immunofluorescence. Decreased levels of PGC‐1α also reduced the CCA‐evoked increases in COXIV. However, PGC‐1α was not necessary for the CCA‐induced changes of all mitochondrial proteins since Tfam and cytochrome c displayed normal increases, regardless of attenuated PGC‐1α levels. Thus, PGC‐1α is necessary for the normal induction of mitochondrial biogenesis, and the coactivator likely functions with parallel pathways to mediate increases in mitochondrial proteins with CCA.
Altered gene expression in the heart after acclimation to hypoxia in goldfish: Evaluation of candidate reference genes for qPCR studies
Authors: Chan Stefanie , Ngo Thanh Thu T. , Cameron John S.
Published: June 2021

Quantitative real‐time PCR (qPCR) studies can be used to investigate altered gene expression in animals exposed to varying environmental conditions, but an accurate analysis requires data normalization with stably expressed internal reference genes. We evaluated the relative stability of five reference genes commonly used in qPCR studies; expression profiles in cardiac muscle were determined after acclimation of goldfish (Carassius auratus) to moderately hypoxic conditions (2.6 mg O2/l) for seven days at 22°C. Transcription levels were quantified for β‐actin (ACTB), ubiquitin (UBI), acidic ribosomal protein (ARP), elongation factor (EF‐1α) and major histocompatibility complex (MHC1); primers for each were designed using previously sequenced genes from goldfish and zebrafish (Danio rerio). Stability of gene expression in acclimated vs. non‐acclimated fish was greatest for ACTB and ARP, while EF‐1α proved unsuitable as a reference, showing a consistent downregulation. Statistical analysis suggested that the combined use of ACTB, ARP and UBI as reference genes would allow a robust interpretation of qPCR data after acclimation to chronic hypoxia. The identification of hypoxia‐stable housekeeping genes in goldfish heart will aid future studies of the molecular mechanisms underlying hypoxia‐ and anoxia‐tolerance in this species. (Supported by the HHMI and the Brachman Hoffman Fund)
Evidence of sternal artery vasoconstriction in Crayfish, Procambarus clarkii (Pc) but not Pacifastacus leniusculus (Pl)
Authors: Violi David William , McClean Brent , Pelaez Nancy
Published: June 2021

Procambarus clarkii (Pc) are known to re‐distribute arterial flows when exposed to hypoxic water. In response to hypoxia, flow to the anterior aorta increases while flows to the posterior dorsal aorta and sternal artery decrease, causing an increase of hemolymph flow to the animal's head. For this study we compared isolated blood vessel function from Pc and another crayfish, Pacifastacus leniusculus (Pl). Both crayfish survive for some time out of water, but Pl is more active physically and has narrower arteries. Sternal arteries from both species were isolated. Compliance was measured in cannulated pressurized blood vessels and isometric force was measured on a wire myograph with bicarbonate‐buffered crayfish saline. After relaxation in a calcium‐free solution containing 0.1 mM EGTA, KCl depolarization produced a small transient contraction for Pc but this response has not been observed for Pl. We are now investigating compliance curves in bicarbonate‐buffered crayfish saline in the presence or absence of calcium for Pc and Pl. Vasoconstriction in arteries may have effects on blood flow resistance in Pc but not in Pl.
Characterization of LPA4 and LPA5 In zebrafish
Authors: Kao Milky Sheng‐Yung , Chang Chig‐Han , Lee Hsin‐Yu
Published: June 2021

Lysophosphatidic acid (LPA) is a simple lysophospholipid which regulates various important biological functions such as cell proliferation, migration, differentiation, and morphogenesis. Multiple receptors for LPA on cell membrane have been identified, designated LPA1‐7. Among these receptors, LPA1‐3 are the members of the endothelial differentiation gene (EDG) family. In contrast, LPA4‐5 are first discovered as purinergic receptors in P2Y family. In zebrafish (Danio rerio), LPA1 and LPA3 were demonstrated to be important regulators for lymphatic vessel and neuronal development, while the roles of other LPA receptors have not yet been identified. As we search LPA receptors by TBLASTN in zebrafish nucleotide database, we found LPA4 and one sequence similar to both mouse LPA5 and P2Y5. After alignment with all known LPA receptors, the sequence shows more similarity with LPA5 than with P2Y5. Our observation suggest that LPA5 gene evolutionally multiplied and differentiate into two receptors. In zebrafish, zLPA4 expressed in gill, testis and spleen, while zLPA5 expressed in most tissues after 18‐somite stage. However, LPA treatment was unable to induce Ca++ mobilization in zLPA4 or zLPA5 transfected B103 rat neuroblastoma, indicating that zebrafish LPA receptors might not trigger conventional signaling cascades of mammalian LPA receptors. Institute of Zoology, National Taiwan Universtiy
Electrical Stimulation Induces AMPK‐Mediated Cell Cycle Arrest in C2C12 Myoblasts
Authors: Duchene Brittany , Connor Michael K.
Published: June 2021

Skeletal muscle is phenotypically dynamic and responds to altered activity. Electrical stimulation (STIM, 3h/day) of differentiated C2C12 myotubes led to 18‐ and 12‐fold increases in the protein levels of p27Kip1 and p21Cip1, respectively. This suggested STIM‐induced cell cycle arrest in these cells. Thus, we stimulated proliferating myoblasts and examined the effects on p27 in these cells. p27 protein levels increased while cyclin E protein levels decreased from one to five days of STIM suggesting these cells underwent cell cycle arrest. Concomitant with these changes was increased phosphorylated AMPK which may underlie these STIM‐induced effects. Similar effects were observed using differentiating C2C12 cells. Marked increases in T198 phosphorylated p27, a direct AMPK stabilizing phosphorylation, were correlated with increased p27 protein levels. Pharmacologic inhibition of AMPK activation completely abolished STIM‐induced increases in AMPK activation, T198 p27 phosphorylation and total p27 protein levels. Thus, electrical STIM alters the activity of AMPK in skeletal muscle cells, induces cell cycle arrest and may mediate cell cycle exit prior to the initiation of differentiation.
PLA2 activity declines during skeletal myoblast differentiation
Authors: Burkholder Thomas J
Published: June 2021

Phospholipase A2 (PLA2) activity contributes to muscle differentiation, regeneration and damage, but the complement of cellular PLA2 expression is poorly defined. Stretch induced differentiation of myoblasts is dependent on prostaglandin F2alpha, a downstream metabolite of PLA2, and stretch induced signaling in myotubes is dependent on PLA2 activity. The PLA2 superfamily include calcium activated cPLA2, calcium independent iPLA2, and small, secreted sPLA2 families. Real time, quantitative PCR was used to determine the expression of cPLA2 alpha‐epsilon and iPLA2 alpha‐zeta in hindlimb muscles and differentiating myoblasts from C57 Bl/6 mice. Greatest expression was found of pla2g4e and pla2g6b, with little difference among muscles. Only pla2g6a showed any correlation with myosin heavy chain isoform. Expression of pla2g4a and c declined ten‐fold during differentiation. Calcium activated PLA2 activity declined in parallel with pla2g4a expression, and calcium‐independent PLA2 activity was not detectable in differentiating myoblasts. These results suggest that PLA2 and eicosanoid signaling within muscle may be highly dependent on cells other than myofibers.

Supported by DC005017
The effects of exercise on skeletal muscle GLUT4 expression in patients with type 2 diabetes
Authors: Yeo Sophie Elizabeth , McGee Sean Leslie , Garnham Andrew , McConell Glenn , Hargreaves Mark
Published: June 2021

The aim of this study was to determine the effects of a single bout of exercise on the regulation of GLUT4 mRNA expression in patients with type 2 diabetes (T2D) compared with healthy control subjects. 9 patients with T2D (54 ± 4 yrs) and 9 control subjects (48 ± 3 yrs) matched for age and BMI performed 60 min of cycling exercise at ~70% VO2peak. Muscle biopsies were obtained at baseline, immediately post and 3 hrs post exercise. For all parameters there was a main effect of time (p<0.05), with no difference between groups. Phospho AMPK (p<0.01) and phospho p38 MAPK (p<0.05) were increased immediately post exercise in control and T2D subjects. Exercise had no effect on phospho CaMKII in either group. Exercise increased GLUT4 mRNA in control (~60%) and T2D (~66%) subjects immediately post exercise, and remained elevated 3 hrs post exercise in both groups (p<0.01). T2D subjects had lower resting PGC1α mRNA compared to controls (p<0.05), and PGC1α mRNA was increased post exercise in both groups. In conclusion, increased signalling through AMPK and p38 MAPK may be a mechanism that contributes to the increase in skeletal muscle GLUT4 mRNA expression following acute exercise. Importantly, exercise mediated regulation of GLUT4 expression is normal in patients with T2D. In this regard, GLUT4, and the signalling proteins that mediate GLUT4 expression, may represent important targets in the treatment of insulin resistance.
The Role of MuRF1 in Glucocorticoid Induced Skeletal Muscle Atrophy
Authors: Baehr Leslie M , Furlow J David , Bodine Sue C
Published: June 2021

Skeletal muscle atrophy occurs under a variety of conditions and is the result of a shift in the balance of protein turnover from net synthesis towards net degradation. Expression of MuRF1, an E3 ubiquitin ligase, is an excellent marker of muscle atrophy and increases under divergent atrophy‐inducing conditions such as denervation and glucocorticoid treatment. While deletion of MuRF1 has been reported to spare muscle mass following 14 days of denervation (Bodine et al. Science 294:2001), its role in other atrophy conditions is unclear. The goal of this study was to determine whether MuRF1 has a more universal role in regulating muscle atrophy, especially under metabolic disorders. The response of the gastrocnemius (GA) and tibialis anterior (TA) muscles to 14 days of dexamethasone (Dex) treatment (3 mg/kg daily) was examined in 4 month old male and female wild type (WT) and MuRF1 knock out (KO) mice. Following 14 days of Dex, muscle wet weight was significantly decreased in the GA and TA of male and female WT mice. Comparison of WT and KO mice revealed significant sparing of mass in the KO mice of both genders. Further, analysis of fiber cross‐sectional area in the GA showed significant sparing in the KO mice compared to WT. These data reveal that MuRF1 has a significant role in atrophy induced by signals other than disuse. In contrast, analysis of MAFbx KO mice under the same conditions showed no muscle sparing effects. This work was supported by NIH RO1 DK75801.
Effects of acute and chronic hypoxia exercise on erythrocyte aggregation and binding of fibrinogen to erythrocyte under shear flow
Authors: Chen Shiang‐Yu , Wang Jing‐Shyan
Published: June 2021

Pathological erythrocyte aggregation reduces capillary perfusion and oxygen transfer to tissue, resulting in tissue ischemia or infraction. This study investigates how hypoxic exercise training affects erythrocyte aggregation and binding affinity of fibrinogen to erythrocyte. Fifteen sedentary healthy men cycled on an ergometer at about 60% of maximal oxygen consumption under 15%O2 condition for 40 minutes per day, 5 days per week for 5 weeks. The erythrocyte aggregation and binding affinity of fibrinogen to erythrocyte were measured by ektacytomety and flow cytometry, respectively. Our results demonstrated that at pre‐training stage, acute 12%O2 exercise increased erythrocyte count, as well as, shortened critical time and heightened critical shear stress in erythrocyte aggregation under conditions of whole blood and the isotonic 1% solution of dextran 500. However, 5 weeks of 15%O2 exercise training attenuated the enhancement of erythrocyte aggregability caused by acute 12%O2 exercise. Additionally, although shear stress disassociated the fibrinogen adhesion to RBC surface in an intensity‐dependent manner, hypoxic exercise did not influence the ability of shear‐disassociated interaction between fibrinogen and erythrocyte. Therefore, we conclude that long‐term moderate‐intensity exercise at 15%O2 environment suppresses erythrocyte aggregation but not changes the binding affinity of fibrinogen to erythrocyte following severe hypoxic exercise.
Systemic hypoxia promotes the mobilization of senescent CD8+ T lymphocytes into the peripheral blood compartment during moderate‐intensity exercise
Authors: Weng Tzu‐Pin , Wang Jong‐Shyan
Published: June 2021

This study investigated how various regimens of exercise with/without hypoxia affected mobilization of T lymphocyte subsets into the peripheral blood compartment. Eighteen sedentary males randomly engaged in two normoxic exercise bouts [severe exercise (SE) (up to VO2max) and moderate‐intensity exercise (ME) (50%VO2max) while exposed to 21%O2], two hypoxic exercise bouts (ME while exposed to 12%O2 and 15%O2) and two hypoxic resting conditions (resting while exposed to 12%O2 and 15%O2) in a normobaric hypoxia chamber. Under normoxic conditions, SE, but not ME, increased the percentages of senescent (CD28− and CD57+)/activated (CD62L− and CD11a+)‐form CD8+T lymphocytes mobilized into the peripheral blood compartment. Although no significant change occurred in the distribution of various CD8+T lymphocyte subsets under resting 12%O2 or 15%O2 condition, the senescent/activated‐form CD8+T lymphocytes markedly released into the blood compartment following ME combined with 12%O2 or 15%O2 exposure. However, the distribution of CD4+T lymphocyte subsets in blood remained unchanged in response to various regimens of exercise with/without hypoxia. We conclude that the 12–15%O2 exposures promote the mobilization of senescent/activated CD8+ T lymphocytes into the peripheral blood compartment during ME.
Effect of passive heating for 14 days on thermoregulatory responses and subjective thermal sensation in humans
Authors: Sato Maki , Kanikowska Dominika , Shimizu Yuuki , Inukai Yoko , Nishimura Naoki , Iwase Satoshi , Sugenoya Junichi
Published: June 2021

We investigated the effect of passive heating on thermoregulatory responses and subjective sensation. Six volunteers were subjected for the experiment at latitude 35°N; in which the mean temperature and hours of daylight were 7.3°C and 11:21 hours in winter. Subjects entered a climatic chamber at 40°C and 50%RH for 90 min for 14 days. Core and skin temperatures, sweat rate and cutaneous blood flow were measured during leg water immersion at 42°C for 30 min in pre‐, 7th and post‐passive heating session in 26°C and 50%RH. Subjective thermal sensation was asked every 5 min during water immersion. Antidiuretic hormone (ADH), angiotensin II, aldosterone, and total proteins were measured in the immersion days. Tympanic temperature, sweat rate and cutaneous blood flow during water immersion were not significantly different between pre‐ and post‐passive heating session. The concentration of ADH, angiotensin II and aldosterone were not significantly different between pre‐ and post‐passive heating session. Thermal sensation rating was significantly lower in post‐ than in pre‐passive heating. These results suggested that subjective thermal sensation might be changed after 14 days passive heating at 40°C and 50%RH without changes in core temperature.
Impaired sweating in multiple sclerosis leads to increased reliance on skin blood flow for heat dissipation
Authors: Davis Scott L. , Korkmas Melissa A. , Crandall Craig G. , Frohman Elliot M.
Published: June 2021

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that can disrupt autonomic function, leading to impaired thermoregulatory function. The aim of this study was to test the hypothesis that control of sweating and skin blood flow (SkBF) is impaired in MS during whole‐body heating (WBH). Sweat rate (SR) and SkBF were assessed in dorsal forearm skin in females diagnosed with MS (n=4, age=39±4 yrs) and matched healthy controls (n=4; age=36±6 yrs) during normothermia (NT) and WBH (increased internal temperature ~1.0 °C). Cutaneous vascular conductance (%CVCmax) was calculated from the ratio of SkBF to mean arterial pressure and normalized to maximal responses obtained by local heating (42 °C for 30 min) following WBH. Sweating (ΔSR from NT) was impaired in MS patients (0.53±0.34 mg/cm2/min) compared to controls (0.99±0.07 mg/cm2/min; P=0.04). Cutaneous vasodilation (Δ%CVCmax from NT) was higher in the MS group (62.6±5.1%) compared to controls (41.6±6.3%; P=0.01). No group differences in cutaneous vasodilation were observed during local heating (P=0.92). Impaired sweating in MS may arise from impairments in neural control of sudomotor pathways or neural‐induced changes in eccrine sweat glands. Interestingly, larger increases in cutaneous vasodilation to WBH suggest neural control of SkBF is intact and may compensate for impairments in sweating in an attempt to adequately dissipate heat in MS patients.

Supported by National MS Society Grant RG4043A1/1
Altered water vapor and evaporation during exercise in a cold weather garment layering system
Authors: Wilkins Brad W , Kvernmo Michelle L , Myhre Loren G
Published: June 2021

Water vapor accumulation within a garment layering system during exercise in the cold may alter evaporative heat loss. The objective of this study was to investigate the impact of altered water vapor accumulation within a garment layering system during cold weather exercise. Subjects (N=10) exercised on a treadmill for 45 min (70% VO2max) while wearing a wind impermeable (air permeability: .02 m3/min/m2) running jacket with (open) and without (closed) open mesh fabric (air permeability: 39 m3/min/m2) panels placed at the chest (120 cm2), sides (120 cm2), and back (120 cm2). Jackets were worn over a standard polyester t‐shirt. Average (front and back) water vapor pressure (Pw) was calculated from humidity (%) and air temperature under the running jacket (between t‐shirt and jacket). Whole body temperature (Twb), total sweat loss, and evaporation of sweat were also determined. Average Pw in the running jacket was 13.1±0.4 and 10.4±0.3 mmHg for the closed and open jacket conditions, respectively. The decreased Pw enhanced evaporation in the open (84±2%) compared to the closed jacket condition (79±2%; P <0.05), without a statistical difference in sweat loss (closed: 654±56 vs. open: 642±55 g) or Twb (closed: 36.6±0.1 vs. open: 36.4±0.2 °C). Results suggest a ~3 mmHg reduction in Pw in running jackets enhances sweat evaporation without a significant reduction in whole body temperature. Research support from Nike Inc.
Treatment with an adenosine receptor antagonist completely blocks fasting‐induced torpor in mice
Authors: Iliff Benjamin William , Swoap Steven John
Published: June 2021

When calorically restricted at cool ambient temperatures, mice conserve energy by entering torpor, during which core body temperature (Tb) and heart rate (HR) decrease. The application of exogenous adenosine produces hypothermia and bradycardia that mimic a torpid state. In this study, we used the non‐specific adenosine receptor antagonist aminophylline to test the hypothesis that adenosine signaling is necessary for the initiation of torpor in fasted mice. Male C57BL/6J mice were infused with aminophylline at a rate of 7 mg kg−1 hr−1 while Tb and HR were continuously monitored using implanted radiotelemeters. Over the course of a 23 hour fast at an ambient temperature of 20°C, all control mice exhibited torpor bouts, reaching a minimum Tb of 26.6 ± 1.1 °C with an average Tb of 32.6 ± 0.8 °C. However, aminophylline‐treated fasted mice did not enter torpor, with a minimum Tb of 34.4 ± 0.7 °C and an average Tb of 37.3 ± 0.2 °C. Likewise, the HR of control fasted mice fell to 153 ± 15 bpm, while aminophylline‐treated mice had a minimum HR of 359 ± 24 bpm over the fasted period. These findings support our hypothesis that adenosine receptor signaling is necessary for the induction of torpor in mice.
Contribution of the paraventricular nucleus in the heat stress‐induced cardiovascular adjustments
Authors: Leite Laura H.R. , Zheng Hong , Coimbra Candido C , Patel Kaushik P
Published: June 2021

We assessed the effect of hyperthermia on sympathetic nerve activity and blood flow redistribution dictated by the paraventricular nucleus (PVN). Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), heart rate (HR), body and tail temperatures were recorded in anesthetized rats after bilateral microinjection of CSF, Lidocaine or L‐NMMA into the PVN during heat stress (n=6/group). Heat stress was induced by graded increase in the temperature of a heating pad for 30 min. This stimulus resulted in blunted RSNA response after PVN blockade with Lidocaine (ΔRSNA: 117.6 ± 17.0 % 11.3 ± 7.3 %, CSF vs. Lidocaine, p<0.05), as well as blunted MAP and attenuation of HR increase (ΔMAP: 22 ± 2 mmHg −0.04 ± 7.2 mmHg; ΔHR: 93.4 ± 9.3 bpm 43.4 ± 18.8 bpm, CSF vs. Lidocaine, p<0.05). Change in body temperature for tail vasodilation was unaffected by Lidocaine treatment. RSNA, HR and MAP in L‐NMMA treated rats increased to heating‐stress, similar to CSF treated rats. However, a higher Δbody temperature until tail vasodilation was observed after L‐NMMA (0.3 ± 0.1 °C 0.8 ± 0.1 °C, CSF vs. L‐NMMA, p<0.01). In summary, the PVN participates in increasing renal sympathetic activity to heating, contributing to cardiovascular adjustments that influence core blood redistribution to the periphery. Furthermore, heating possibly increases heat loss through peripheral vasodilation via a nitric oxide mechanism within the PVN.
Electroencephalographic effects of RVMM inhibition in free‐behaving rats
Authors: Tupone Domenico , Cerri Matteo , Luppi Marco , Martelli Davide , Mastrotto Marco , Amici Roberto
Published: June 2021

Decrease in core temperature during torpor is concomitant with a left shift in the peak of the theta band of the EEG (5,5–9 Hz). It has been shown that inhibition of rostral ventromedial medulla (RVMM), produces a deep hypothermia in free behaving rats, caused by a massive peripheral vasodilation, and a concomitant inhibition of thermogenesis. The aim of our study was to investigate if the hypothermia obtained by RVMM inhibition in free behaving rat (that does not display spontaneous episode of torpor) also produced a shift in the theta frequency of the EEG. Eleven male CD Sprague‐Dawley rats were used. Animals were implanted under general anaesthesia with: electrodes for EEG recording, an hypothalamic thermistor to record deep brain temperature and a microcannula in RVMM for the drugs microinjection. Following 7 days of recovery, experiments were performed in a thermoregulated and sound‐attenuated box. After 2‐h baseline recording, animals were injected in RVMM with 100 nl of the following: a) 1 mM GABA‐A agonist muscimol b) isotonic saline (NaCl 0.9%). Muscimol evoked a rapid cutaneous vasodilation followed by a deep hypothermia and by a left shift in the theta band. These data shown that the theta band of the EEG is left‐shifted after centrally induced hypothermia, even in animals that don't display episodes of torpor. Supported by MUR, Italy, University of Bologna, and ESRS (Sanofi‐Aventis Research Grant).
The blockade of oxytocinergic receptors in the CeA modify VP plasma levels and mRNA expression induced by hypertonic BVE in the rat.
Authors: Margatho Lisandra O , Elias Carol F , Elias Lucila LK , Antunes‐Rodrigues Jose
Published: June 2021

We investigated the effects of oxytocin (OT) and the oxytocinergic antagonist vasotocin (OVT) injection in the central amygdala (CeA) in the regulation of vasopressin (VP) mRNA expression in the hypothalamus and VP plasma levels in response to hypertonic blood volume expansion (hyper BVE). Male Wistar rats with cannulas unilaterally in the CeA were subjected to BVE (2 ml/100 g bw) over 1 minute. OT, OVT or vehicle was injected into the CeA 20 min before the BVE. Five min after BVE rats were decapitated and the blood was collected for VP plasma determination. Another group was perfused 40 min after BVE. Brain sections of the paraventricular (Pa) and the supraoptic (SON) nuclei were collected for in situ hybridization using a 35S‐labeled VP probe. OT injection in the CeA increased plasma VP levels after hyper BVE and reduced VP mRNA in the PaLM. OVT reduced plasma AVP levels following a reduction VP mRNA in the PaLM and SON. Our data suggest that OT in the CeA may modulate the physiological mechanisms involved in plasma AVP release but the time course of the hormonal release has been shown dissociated from the genomic AVP expression in the PaLM. We hypothesize that the blockade of OT CeA receptors with OVT may activate an inhibitory input to vasopressinergic magnocellular neurons in the hypothalamus leading to the decrease in plasma AVP levels that can be strongly correlated with the reduction in the genomic AVP mRNA.
Oxidative stress and androgens have a synergistic effect on dopamine cell viability
Authors: Cunningham Rebecca L , Singh Meharvan
Published: June 2021

Parkinson's disease (PD) is a progressive neurodegenerative condition associated with oxidative stress and dopamine neuronal loss in the nigrostriatal pathway. Aged men have a greater incidence of PD than women. While estrogens have been shown to play a neuroprotective role in PD, there is little information on androgens’ actions on dopamine neurons. In this study, we examined if androgens alter the effects of oxidative stress in a dopaminergic cell line (N27 cells). N27 cells were exposed to tert‐butyl‐hydrogen peroxide (500uM) 24hrs prior to androgen treatment (24hr). Our results show that hydrogen peroxide sensitized N27 cells to testosterone‐induced cell death, as indicated by a decrease in calcein fluorescence. Thus, the degree of oxidative burden influences the effects of androgens on cell viability. Interestingly, androgens did not alter cell viability when cells were concurrently exposed to hydrogen peroxide and testosterone. Thus suggesting increased oxidative stress, as associated with aging, is an integral component of androgens’ effects on dopaminergic cell viability. These results indicate that basal oxidative stress levels modulate the influence of androgens on dopamine neuronal viability. Supported in part by NIH grants F32NS061417‐01 to RLC and AG022550 and AG027956 to MS.
Nesfatin‐1 Influences the Excitability of Neurons in the Nucleus of the Solitary Tract
Authors: Mimee Andrea V , Hoyda Ted D , Ferguson Alastair V
Published: June 2021

Nesfatin‐1 is a recently discovered neuropeptide that has been shown to decrease food intake in animals when injected both intracerebroventricularly and peripherally. In situ hybridization and immunohistochemical studies have revealed the expression of nesfatin‐1 throughout the brain and, in particular, in the nucleus of the solitary tract (NTS), a medullary structure with important roles in the regulation of feeding. In order to shed light on the mechanisms by which nesfatin‐1 exerts its satiety‐promoting actions, this study examined the effect of nesfatin‐1 on the excitability of NTS neurons. Whole‐cell current clamp recordings from rat NTS neurons in slice preparation were performed and the effects of bath application of nesfatin‐1 (10nM) on membrane potential examined. Of 14 cells tested, 43% (6/14) were influenced by nesfatin‐1. Depolarizing effects were seen in 50% of responsive cells (mean depolarization = 5.1 ± 1.4 mV, n=3) while the remaining 50% of cells hyperpolarized (mean hyperpolarization = −8.6 ± 0.9 mV, n=3) in response to nesfatin‐1. These findings identify the NTS as a potential site at which nesfatin‐1 acts to influence central control of food intake.

Research support provided by NSERC, CIHR, and HSFC.
Gastric infusion of nutritional supplement in fasted rats increases splanchnic sympathetic nerve activity (sSNA) and activates RVLM‐projecting neurons of the paraventricular hypothalamus (PVH)
Authors: Sharpe Amanda Lea , Andrade Mary Ann , Herrera‐Rosales Myrna , Calderon Alfredo S , Toney Glenn M
Published: June 2021

The hypothesis that refeeding of fasted rats would stimulate SNA via activity of sympathetic regulatory neurons in the PVH was tested. In one study, immunohistochemistry was used to determine PVH neurons stimulated by “refeeding” (Ensure, 10 ml, intragastric) in 3 day fasted rats previously injected with the retrograde tracer cholera toxin subunit B (CTB) into the rostral ventrolateral medulla (RVLM). Sympathetic regulatory neurons (CTB‐immunofluorescence, CTB‐IF), neurons activated by refeeding (c‐Fos‐IF) and double labeled neurons were counted in 3 rostro‐caudal anatomically matched sections from the PVH of each rat. Refeeding increased c‐Fos‐ IF in the PVH compared to control rats. Whereas CTB‐IF and c‐Fos‐IF neurons were located throughout the PVH, c‐Fos‐IF was higher in the rostral PVH, and CTB‐IF neurons were more prevalent in the medial and caudal portions of the PVH. Colocalization of CTB and c‐Fos were noted in approximately 12% of CTB‐IF neurons. In a second study, sSNA was recorded in anesthetized, 72 h fasted rats at baseline and following intragastric delivery of Ensure (10 ml). sSNA increased in a slow, linear fashion to approximately 140% of baseline at 1 hour post refeeding. Together these results suggest that sympathetic regulatory neurons in the PVH may mediate the increase in sSNA following refeeding after fasting. Support: HL088052, HL076312 (GMT)
Sitagliptin lowers glucagon and improves glucose tolerance in prediabetic obese SHROB rats
Authors: Ernsberger Paul , Chen Brian , Hou Diana , Escobedo Liza , Moore Alexandra , Koletsky Matthew S , Koletsky Richard J
Published: June 2021

Inhibitors of dipeptidyl dipeptidase IV (DPP‐IV) are effective hypoglycemic agents in type 2 diabetes through potentiation of incretin hormones that act in the pancreas to increase insulin and decrease glucagon release. We sought to determine whether the DPP‐IV inhibitor sitagliptin might be effective in prediabetes relative to standard therapy with the sulfonylurea glyburide, by using the spontaneously hypertensive obese (SHROB) rat model. SHROB show normal fasting glucose but are insulin resistant and hyperglucagonemic. SHROB were treated for 6 wk with vehicle, sitagliptin (30 mg/kg/d) or glyburide (1 mg/kg/d). Body weight, food intake and fasting glucose were all unchanged in all 3 groups, but glucagon was reduced 33% by sitagliptin while unchanged following glyburide or vehicle. In oral glucose (6g/kg) tolerance testing, both sitagliptin and glyburide lowered plasma glucose. Both sitagliptin and glyburide shifted peak insulin secretion earlier (30 min for glyburide and 60 min for sitagliptin but 240 min for vehicle). Only sitagliptin significantly enhanced insulin secretion. Sitagliptin is effective in normalizing excess glucagon levels and delayed exaggerated insulin secretion in response to a glucose challenge in a prediabetic model.
Age‐related anabolic resistance after endurance type exercise in humans
Authors: Durham William , Casperson Shanon , Dillon Edgar Lichar , Keske Michelle A , Paddon‐Jones Douglas , Sanford Arthur , Lakshman Kishore M , Hickner Robert C , Hong Chuan , Grady James , Sheffield‐Moore Melinda
Published: June 2021

Age‐related skeletal muscle loss is thought to stem from suboptimal nutrition and resistance to anabolic stimuli. Impaired microcirculatory (nutritive) blood flow may contribute to anabolic resistance by reducing delivery of amino acids to skeletal muscle. In this study, we employed contrast‐enhanced ultrasound and microdialysis sampling of skeletal muscle interstitium, along with stable isotope methodology, to assess hemodynamic and metabolic responses of older individuals to endurance type (walking) exercise during controlled amino acid provision. We hypothesized older individuals would exhibit reduced microcirculatory blood flow, interstitial amino acid concentrations, and amino acid transport when compared with young controls. We report for the first time that aging induces anabolic resistance following endurance exercise, manifested as reduced (by ~40%) efficiency of muscle protein synthesis. Despite suggesting reduced nutritive flow in the old, our findings comprehensively refute our hypothesis that amino acid availability limits post‐exercise anabolism in older individuals, as circulating and interstitial amino acid concentrations and phenylalanine transport into skeletal muscle were all equal or higher in older individuals than in the young. Our data point to alternative mediators of age‐related anabolic resistance and importantly suggest correction of these impairments may reduce requirements for and increase the efficacy of dietary protein in the old.
Differential gene expression of Toll‐like receptors in response to muscle injury in young and old muscles
Authors: Machida Shuichi , Kunisawa Yumi , Peake Jonathan
Published: June 2021

Toll‐like receptors (TLRs) are highly conserved trans‐membrane proteins that function as pattern recognition receptors for a wide variety of infectious micro‐organisms. Recent research has indicated that TLRs are expressed in skeletal muscle. The purpose of this investigation was to determine the mRNA expression pattern of six TLRs during muscle regeneration after muscle injury in young and old muscles. To induce local muscle injury, cardiotoxin was injected into the tibialis anterior muscle of young (2 months) and old (28 months) male C57BL/6 mice. The muscles were removed 1, 3, 5, 7, and 14 days after injection. Gene expression of TLR2, TLR4, TLR5, TLR7 and TLR9 was significantly upregulated in muscle from both young and old mice after muscle injury. TLR2 mRNA expression in young muscle peaked after 1 day at a significantly higher level compared with old muscle, and then declined progressively. The expression pattern of TLR4 and TLR9 mRNA was similar in muscle from young and old mice. TLR5 mRNA expression peaked after 1 day in young mice, whereas it did not increase until after 3 days in old mice. TLR7 mRNA expression peaked after 3 days in young mice at a significantly higher level compared with old mice. These data suggest dysregulation of TLR mRNA expression may partially account for the impaired muscle regeneration of aging skeletal muscle.
D2O to determine muscle protein synthesis rates in response to post‐exercise nutrition in adults
Authors: Robinson Matthew , Turner Scott , Hellerstein Marc , Miller Benjamin
Published: June 2021

Muscle protein synthesis (MPS) is acutely stimulated by either endurance exercise or protein consumption, but the long‐term and combined effects in older adults are unknown. We used deuterium oxide (D2O) to evaluate long‐term effects of post‐exercise nutrition and endurance training on MPS. Sedentary adults (n=16, 50±8 yrs) performed 6‐wks of endurance training and consumed either a carbohydrate (CHO) or carbohydrate and protein (PRO) drink after exercise. D2O (50 ml tid) was administered during the training period to maintain body water pool enrichment at 1–2%. VO2 max was measured pre‐ and post‐training as a surrogate for mitochondrial function. MPS over the 6‐wks was determined from total percipitable protein in a 10–20 mg muscle biopsy by gas chromatography‐mass spectroscopy. Training increased (p<0.05) VO2 max in the PRO (25.5 ± 4.2 to 28.1±3.9 ml/kg/min) but not in CHO group (31.1 ±7.1 to 32.5 ±9.1 ml/kg/min). MPS was not different between CHO and PRO groups (0.06 ± 0.004 vs. 0.05 ±0.003 %/day, p=0.23). Although MPS was not different between CHO and PRO, the VO2 max increase with PRO suggests post‐exercise nutrition may impact aerobic training adaptations. Further analysis will evaluate long‐term mitochondrial proliferation. Our use of D2O provides novel insight into long‐term synthesis rates in aging humans. Funding from The Colorado Nutritional Research Unit.
Oxidative stress leads to a decline in mitochondrial capacity in aged fast‐twitch, but not slow‐twitch mouse muscle
Authors: Marcinek David Joseph , Villarin Jason , Kruse Shane
Published: June 2021

Reactive oxygen species (ROS) are implicated in cell pathology associated with many degenerative diseases, but are also important signaling molecules involved in adaptive responses in cell energy metabolism. Our goal was to determine how age and muscle fiber type influence the response of skeletal muscle to a given oxidative stress. We used paraquat (PARA) treatment to induce acute oxidative stress in vivo in young (4 month old) and old (27 month old) mice. To examine how this response varies with muscle fiber type we measured mitochondrial metabolism in permeabilized soleus (SOL, slow‐twitch) and extensor digitorum longus (EDL, fast‐twitch) muscles. In the EDL of old mice there was a significant (24%) decline in state 3 respiration one week after treatment with 30 mg/kg PARA, while in young mice PARA treatment resulted in a trend toward an increase (20%) in state 3 respiration. In contrast, there was no effect of PARA treatment on mitochondrial respiration in young or old SOL. Our results indicate that fast‐twitch muscle is more susceptible to oxidative stress induced changes in mitochondrial metabolism and that these effects are qualitatively different in young and old muscle. Supported by NIH grants AG028455, AG022385, AG029052 and the Ellison Medical Foundation.
Claudin‐4 is required for EGFR‐mediated alveolar epithelial migration
Authors: Rokkam Deepti , Ho Gwendolyn , Frank James A.
Published: June 2021

Claudin‐4 is tetraspan membrane protein known to play a role in paracellular conductance at tight junctions. Recent studies have shown an uncharacteristic diffuse localization of this protein in epithelial injury and this change in protein trafficking remains unexplained. It is our hypothesis that claudin‐4 plays a role in response to injury. To test this hypothesis, we utilized an alveolar epithelial type 2 cell scratch‐wound healing model. Claudin‐4 expression increased significantly with wounding whereas other claudins remained unchanged. Claudin‐4 expression was higher only in cells along the wound edge, and the protein was widely distributed in the cells. This change in trafficking was unique to claudin‐4 compared with other claudins and zona occludens‐1 (ZO‐1). Inhibition of claudin‐4 with a blocking peptide or siRNA delayed wound healing, indicating a potential role in repair. Since previous studies have shown claudin‐4 is induced by epidermal growth factor (EGF), we studied the activation of its receptor (EGFR) in this model. EGFR was activated along the wound edge in a pattern similar to the increase in claudin‐4. Inhibition of EGFR by AG1478 blocked the increase in claudin‐4 and delayed wound healing comparably to inhibition of claudin‐4 alone. We propose that expression of claudin‐4 is locally induced in wounded epithelia by EGFR and that claudin‐4 plays a novel role in EGFR‐mediated wound healing.
Design elements of a longitudinal course in human structure
Authors: Metzger Keith , Gannon Patrick , Rennie William
Published: June 2021

Human structure, defined here as a spectrum of relationships from cellular to gross, is traditionally taught as a series of relatively short isolated courses that may or may not be integrated. At our School of Medicine, topics encompassed under the umbrella of structure include gross anatomy, embryology, histology, gross/histopathology and clinical imaging. These topics are presented within a single, lab‐based course spanning the first 100 weeks of the four year curriculum. The “Structure” course emphasizes the integration of basic science and clinical concepts in an active learning environment. Learning sessions within the longitudinal course are themselves integrated within each 9–12 week systems‐based course.

Design of the Structure course involved a significant degree of input from clinicians who serve as primary facilitators for student learning. Presentation of topics includes: 1) broad primary exposure with subsequent expansion of depth and complexity, 2) an internally integrated organization, in which multiple aspects of individual structural topics are presented in a single session, and 3) use of active pedagogies including small‐group learning and problem‐based dissection. It is our expectation that this approach will facilitate the advancement of students through increasing levels of cognition, promote retention and enhance the clinical application of structural topics.