bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrophysiological properties of enkephalinergic neurons visualized in BAC transgenic mice
Authors: Tsuda Masayuki , Takasusuki Toshifumi , Hori Yuuichi
Published: June 2021

Enkephalinergic neurons have been found in several areas of the central nervous system, including the spinal cord dorsal horn, where they play an important role in the transmission and modulation of nociceptive information. In an attempt to visualize enkephalinergic neurons in vivo, we generated a Bacterial Artificial Chromosome transgenic mouse in which enhanced green fluorescent protein (EGFP) is expressed in enkephalinergic neurons under the control of the preproenkephalin gene promoter. Immunohistochemical studies indicated that the expression pattern of EGFP is similar to that of endogenous enkephalin.

Patch‐clamp recordings were made from EGFP‐fluorescent neurons located in the superficial dorsal horn of spinal cord slices, and the electrophysiological properties of these neurons were investigated. Some neurons exhibited bursts of action potentials throughout a depolarizing current pulse, other neurons showed a strong accommodation of action potentials, and other neurons fired a single action potential in response to a depolarizing current pulse. The remaining neurons showed different firing patterns. EGFP‐fluorescent enkephalinergic neurons in the superficial dorsal horn come in a variety of electrophysiological properties.

This work was supported by grants from the Promotion and Mutual Aid Corporation for Private Schools of Japan (MT).
Effects of Caffeine on Exercise Fatigue in Sleep Deprived Mice
Authors: Zielinski Mark R. , Carmichael Martin D. , Murphy E. Angela , Youngstedt Shawn D. , Davis J. Mark
Published: June 2021

In humans, exercise run‐time to fatigue is reduced following sleep deprivation. Caffeine can be effective in reducing fatigue in many conditions, but there is no evidence regarding the effect of caffeine on run‐time to fatigue following sleep deprivation. We have developed an animal model of sleep deprivation and fatiguing exercise in order to better address potential related CNS mechanisms. Twenty‐four male CD‐1 mice were randomly assigned to 4 conditions in a 2 SLEEP (normal sleep or sleep deprivation) X 2 SUPPLEMENT (caffeine or placebo) experimental design. Sleep deprivation groups were placed on a slow rotating disc for 24 h to prevent sleep. Normal sleep groups were concurrently placed on the same apparatus, but with the disc locked so that sleep was allowed. Mice received an i.p. injection of either caffeine (6 mg/kg) or placebo (saline) 30 min prior to exercise. Immediately after sleep deprivation or normal sleep, mice ran on a treadmill (24 m/min) until fatigue. Then, the mice were sacrificed and plasma was collected for glucose analysis. Sleep deprivation significantly reduced run‐time to fatigue (p < 0.05) and caffeine attenuated this effect (p < 0.05). No significant treatment differences in plasma glucose were found. Further research is planned to address potential CNS mechanisms in this animal model.
Quantification of the cortical silent period evoked via transcranial magnetic brain stimulation
Authors: Clark Brian C , Hoffman Richard L. , Damron Leatha A.
Published: June 2021

A magnetic pulse to the motor cortex during a muscle contraction produces a motor evoked potential (MEP) followed by electrical quiescence before activity resumes (cortical silent period [CSP]). The CSP is used clinically and scientifically despite little consensus on how to best assess and quantify it. In this study we assessed the stability and reliability of different CSP quantification methods by recording the CSP from 7 humans at 3 stimulus intensities (10, 20 and 30% active motor threshold [AMT]) and quantifying it based on 5 commonly used onset points (see figure). Inter‐rater reliability of CSP quantification was high (r≥0.95). The coefficient of variation (CV) was reduced when the reference point used to demarcate the CSP onset was earliest in the temporal occurrence of events (range 14–33%). The lower stimulus intensities (10 and 20% AMT) exhibited high CV's (>20%), and random error (LOA Ratio: 73–98), whereas the amount of variability evident at 30% was not associated with the length of the CSP (systematic bias <1‐ms). These results suggest that CSP studies should utilize a stimulus intensity ≥30% AMT and define the onset of the CSP at the MEP onset.
Acid‐sensing ion channel‐1a (ASIC1a) contributes to fear and associated autonomic/cardiovascular responses
Authors: Ziemann Adam E , Sabharwal Rasna , Welsh Michael J , Chapleau Mark W , Wemmie John A
Published: June 2021

Understanding fear mechanisms may suggest new therapies for anxiety and its cardiovascular sequela. Previous studies suggest that ASIC1a is enriched in fear circuit structures and is critical for fear behavior. Here we tested the hypothesis that ASIC1a mediates both behavioral and cardiovascular responses to fear. Arterial blood pressure and heart rate (HR) were recorded by radio telemetry in conscious wild type (n = 3) and ASIC1a‐null mice (n = 4) before and during exposure to the fear‐evoking predator odor trimethylthiazoline (TMT) from foxes. Fear‐related freezing behavior was quantified from video recordings. Baseline mean arterial pressure (MAP) and HR did not differ between genotypes. In wild‐type mice, TMT‐induced freezing was accompanied by a reduction in HR and MAP; HR decreased from 795 ± 6 (−TMT) to 374 ± 28 beats/min (+ TMT), and MAP fell from 155 ± 11 to 88 ± 6 mmHg (P < 0.05). Blocking peripheral muscarinic receptors with methylscopolamine reduced TMT‐evoked bradycardia and hypotension, but not freezing. A control odor (butyric acid) failed to evoke freezing, bradycardia or hypotension. In ASIC1a‐null mice, the effects of TMT on freezing, HR, and MAP were attenuated. We conclude: 1) fear‐evoked freezing is accompanied by a parasympathetic‐mediated decrease in HR and a decrease in MAP, and 2) ASIC1a is critically involved in behavioral and autonomic/cardiovascular fear responses (AHA 0760046Z).
Hypertension in the diet induced obese rat is independent of body weight gain
Authors: Zhao Huawei , Northcott Carrie A , MohanKumar Puliyur , Mohankumar Sheba , Fink Gregory D , Haywood Joseph R
Published: June 2021

This study tested the hypothesis that the elevated mean arterial pressure (MAP) observed in the diet induced obese rats (DIO) on normal chow diet is associated with early life weight gain. A radiotelemetry transmitter was implanted in the femoral artery of 4 week old DIO and diet resistant (DR) rats for measurements of MAP and heart rate (HR) over a four week period. All rats were maintained on a normal diet. At 5 weeks, MAP was higher in DIO vs. DR rats (97±1 vs. 89±1 mm Hg, p<0.05), while body weight was not different (DIO: 133±5 gms and DR: 127±2 gms, p>0.05). At 9 weeks, MAP was still significantly elevated (DIO: 114±1 mmHg vs. DR: 103±1 mmHg), but the DIO rats weighed more than the DR rats (336±12 vs. 300±6 gms, p<0.05). At 9 weeks, there was no difference in plasma renin activity (4.0±0.6 vs. 3.5±0.6 ng/ml/hr, p>0.05), aldosterone (82±24 vs. 55±19 pg/ml, p>0.05) and leptin (3.0±0.2 vs. 3.4±0.4 pg/ml, p>0.05). MAP response to ganglionic blockade was similar between the two groups of rats (−43±2 vs. –37±2 mmHg). However, plasma insulin was significantly elevated in the DIO compared to the DR rats (DIO: 1.9±0.2 pg/ml vs. DR: 1.5±0.1 pg/ml). In conclusion, hypertension in DIO rats precedes an enhanced weight gain suggesting that genetic factors predetermine the hypertensive phenotype independent of body weight.
Monocytes stimulate replication of human subcutaneous preadipocytes and reduce adipocyte differentiation
Authors: Couturier Jacob , Lewis Dorothy , Balasubramanyam Ashok
Published: June 2021

HIV Lipodystrophy is a condition identified mainly by adipose tissue redistribution and metabolic abnormalities. Persons with HIV Lipodystrophy have higher numbers of leukocytes, such as monocytes and lymphocytes within their adipose tissues compared to controls, indicating that immune cells may have a role in the adipose tissue changes. However, evidence is lacking that describe interactions between immune cells and adipose cells. To determine if leukocytes can influence adipose tissue growth and morphology, human PBMC's such as lymphocytes or monocytes were co‐cultured with human subcutaneous preadipocytes for up to 6 days. Utilizing cell cycle analysis with flow cytometry, monocytes were found to increase preadipocyte replication (percentages in the S+G2/M phase) 2–3 fold compared to controls, whereas addition of lymphocytes had no effect. In addition, when preadipocytes were then induced to differentiate into adipocytes, the ability to mature into lipid‐filled adipocytes was reduced by 60% in preadipocytes incubated with monocytes as measured by Oil Red O staining for triglyceride accumulation. Although high amounts of inflammatory cytokines such as IL‐6 and IL‐8 were found in the co‐culture supernatants and despite reports showing that IL‐6 and IL‐8 can stimulate preadipocyte proliferation, the use of neutralizing abs against IL‐6 and IL‐8 during our co‐culture experiments failed to block the enhanced replication of the preadipocytes suggesting the involvement of different molecules. These findings suggest that immune cells may be involved in the adipose tissue alterations observed not only in HIV Lipodystrophy but other complications involving immune cell infiltration of adipose tissues such as Obesity and other inflammatory conditions.
Activation of glucagon receptor signaling stimulates regulators of hepatic fat oxidation in vivo
Authors: Berglund Eric D. , Kang Li , Lee‐Young Robert S. , Lustig Daniel G. , Lynes Sara E. , Donahue E. Patrick , Wasserman David H.
Published: June 2021

Evidence suggests that glucagon (GN) has persistent effects on the liver that we hypothesize are related to AMPK, PPARα, and FGF‐21. These proteins are known to promote hepatic oxidative processes. We aimed to define signaling pathways downstream of the hepatic GN receptor in vivo. The role of GN stimulation was tested using wild type (WT) and glucagon receptor null (KO) mice catheterized in a carotid artery and jugular vein 5d prior to study. In 5h fasted mice, GN (10ng/kg/min) or saline (SAL) was infused for 2h and the interaction of GN with circulating fat levels was tested in separate studies in which intralipid (IL) was also infused. Pfloridzin was infused in all studies to prevent high glucose and insulin levels. Euglycemia (150mg/dL) was maintained using a variable glucose infusion. Elevated GN (∼5‐fold) in WT increased glucose production and caused increases in AMP:ATP (7.7±0.5 fold), p‐AMPK (2.1±0.3 fold), and PPARα and FGF‐21 mRNA (2.0±0.3 and 2.0±0.4 fold) in liver compared to SAL. IL alone had no effects. However, IL led to marked potentiation of GN‐induced stimulation of FGF‐21 mRNA (12.3±4.2‐fold) in WT mice. Effects of GN with and without IL were eliminated in KO mice. We show in vivo that glucagon drops the liver to a low energy state which corresponds to increased activation of AMPK and induction of PPARα and FGF‐21 mRNA. Effects of GN on FGF‐21 are potentiated by IL. In conclusion, GN not only acutely regulates glucose homeostasis, but also causes adaptations in processes that improve the ability of the liver to oxidize fat. Supported by DK 50277.
Structural Remodeling of Nucleus Ambiguus (NA) Projections to Cardiac Ganglia in Transgenic Type 1 Diabetic Mice (OVE26)
Authors: Huang Chenghui , Li Lihua , Shang Xinyan , Epstein Paul N. , Cheng Zixi (Jack)
Published: June 2021

Baroreflex control of heart rate is impaired in OVE26 mice. However, the location and the nature of such a change remain unclear. OVE 26 diabetic and FVB control mice (6 months, n = 6/group) received Fluoro‐Gold (ip) injection to label cardiac ganglia and DiI microinjection into the left NA to label vagal efferent. A confocal microscope and a Neurolucida 3‐D digitization system were used to examine vagal efferent axons and terminals in cardiac ganglia. Compared to FVB, we found in OVE26 mice that: (1) the size of cardiac ganglia was reduced (FVB: 0.14±0.01 mm2 vs. OVE 26: 0.11±0.01 mm2, p<0.01, n = 20 ganglia). Somatic area of cardiac principal neurons (PNs) was reduced (FVB: 422±12 μm2 vs. OVE 26: 372±10 μ m2 p<0.01, n = 180 PNs). The density of PNs in ganglia was increased (FVB: 1325±52/mm2 vs. OVE26: 1753±62 /mm2, p<0.01, n = 23 ganglia). (2) NA axons innervated PNs with basket endings. The percentage of the PNs innervated by DiI‐labeled axons in ganglia was decreased (FVB: 49.1±1.6% vs. OVE26: 30.6 ± 1.8%, p<0.01, n = 23 ganglia). The number of vagal axonal varicosities around PNs was decreased (FVB: 68 ± 2/PN vs. OVE26: 32 ± 1/PN, p<0.05, n = 50 PNs). Thus, diabetes significantly changed the structure of cardiac ganglia and reduced vagal efferent projections to cardiac ganglia. Our data may provide a new insight for diabetes‐induced cardiac neuropathy. *The two authors equally contributed to this work. NIH R01 HL‐79636.
Superoxide scavenging in the paraventricular nucleus (PVN) reduces sympathoexcitation and improves cardiac function following myocardial infarction
Authors: Infanger David W , Braga Valdir A , Stupinski John A , Sharma Ram V , Davisson Robin L
Published: June 2021

We have shown that forebrain oxidative stress is involved in the increased sympathetic output that accompanies myocardial infarction (MI)‐induced heart failure in mice. Here we hypothesized that removal of superoxide from the PVN, a brain region controlling neurohumoral drive, would normalize sympathetic output and improve cardiac function following MI. Male C57BL/6 mice received dual PVN injections (200nL) of adenovirus encoding copper‐zinc superoxide dismutase (CuZnSOD, 1×1011 pfu/mL) or control β‐gal (0.9×1011 pfu/mL), followed by ligation of the left anterior descending artery to cause MI. Heart function was assessed by high resolution echocardiography and sympathetic output by norepinephrine (NE) ELISA 14 days later. MI elevated PVN superoxide levels (DHE fluorescence 1.98 fold sham ± 0.21, p<0.01, n=10) which were normalized by CuZnSOD overexpression (1.09 ± 0.14, p<0.01, n=10). MI decreased ejection fraction (29.1 ± 4.2% vs sham 54.8 ± 3.7%, p<0.01, n=16) and increased left ventricle end‐volume (87.7±6.5 vs sham 48.1±2.9 μL3, p<0.01, n=16) which were restored by CuZnSOD (52.3 ± 4.3%, 50.7±3.3 μL3, p<0.01, n=11). NE levels were normalized in CuZnSOD‐treated mice (sham 200±24, MI 491±42, MI CuZnSOD 354±37 ng/mL, p<0.05, n=10–11), implicating oxidative stress in PVN in increasing sympathetic drive after MI. These data support that ROS scavenging in the PVN can improve cardiac function following MI.
Exercise training normalizes enhanced peripheral chemoreflex function in chronic heart failure rabbits
Authors: Li Yu‐Long , Ding Yanfeng , Agnew Chad , Schultz Harold D.
Published: June 2021

Our previous studies have shown that the enhanced sensitivity of the peripheral chemoreflex contributes to the sympathetic hyperactivity in chronic heart failure (CHF) rabbits and an augmented carotid body (CB) chemoreceptor activity is involved in the enhancement of peripheral chemoreflex function. Furthermore, we have also found that upregulation of angiotensin (Ang) II‐AT1 receptor pathway and downregulation of NOS‐NO pathway in the CB contribute to the enhanced CB chemoreceptor sensitivity in the CHF state. Here we investigated whether exercise training (EXT) normalizes the enhanced peripheral chemoreflex function in CHF rabbits and possible mechanism mediating this effect. EXT partially but not fully normalized the exaggerated baseline renal sympathetic nerve activity (RSNA) and the response of RSNA to hypoxia in CHF rabbits. EXT also decreased the baseline carotid body nerve single‐fiber discharge (4.9 ± 0.4 vs 7.7 ± 0.4 imp/s at PO2 =103 ± 2.3 mm Hg) and the response to hypoxia (20.6 ± 1.1 vs 36.3 ± 1.3 imp/s at PO2 =41 ± 2.2 mm Hg) from CB chemoreceptors in CHF rabbits. Western and immunofluorescence data showed that protein expression of nNOS was increased in the CBs from EXT + CHF rabbits, compared with that in CHF rabbits. On the other hand, the AT1 receptor overexpression of the CB in CHF state was also blunted by EXT. These results indicate that EXT improves the enhanced chemoreflex in CHF, which is due to EXT‐induced upregulation of nNOS‐NO pathway and downexpression of Ang II‐AT1 receptor pathway in the CBs.
High salt intake deteriorates cardiac function of hypertrophied heart, possibly via excessive sympathetic drive
Authors: koji Ito , Hirooka Yoshitaka , Sunagawa Kenji
Published: June 2021

Background: High salt intake often results in congestive heart failure in patients with left ventricular hypertrophy (LVH). We hypothesized that excessive sympathetic activation associated with salt intake plays a significant role in developing cardiac dysfunction.

Methods and Results: We used ICR mice and applied aortic banding (AB) to create LVH. Four weeks after the banding, 24‐h urinary norepinephrine excretion (U‐NE) was higher in AB mice than in controls. LV function evaluated echocardiographically, however, was not different between groups. We then fed the mice a high salt diet (8%) for 4 weeks. LV function decreased in the AB mice compared with controls and was significantly worse in the high salt (HS)‐AB mice than in regular salt (RS)‐AB mice (LVDD: 3.5±1 vs 3.2±2 mm, wall thickness: 1.4±0.3 vs 1.2±0.1 mm, fractional shortening: 30±4 vs 44±1 %, all p<0.05). U‐NE was higher in HS‐AB than in RS‐AB mice. Further, the mice fed a high salt diet concurrent with AB developed LVH after 4 weeks, but LV systolic function remained normal without increases in U‐NE.

Conclusions: In mice with LVH, high salt intake deteriorates cardiac function possibly through sympathetic overactivation. These results suggest that sympathetic activation plays a key role in the development of cardiac dysfunction.
Chronic blockade of central V1 receptors reduces resting blood pressure and cardiovascular responses to alarming stress in the infarcted rats subjected to chronic stress
Authors: Szczepanska‐Sadowska Ewa Krystyna , Dobruch Jakub , Cudnoch‐Jedrzejewska Agnieszka , Gomolka Ryszard , Puchalska Liana
Published: June 2021

Previous studies have shown that short‐lasting blockade of central V1 receptors (V1R) reduces mean arterial blood pressure (MABP) and MABP and heart rate (HR) responses to air jet stress in the infarcted rats. The purpose of the present study was to determine effects of chronic stressing (4 weeks) on resting MABP and HR and on cardiovascular responses to air jest stress in the infarcted rats subjected to chronic left ventricle (LCV) infusions of either V1R antagonist or saline. After stressing rats infused with V1R antagonist manifested significantly lower resting MABP than those receiving saline. Pressor and tachycardic responses to air jet stress were significantly lower during infusion of V1R antagonist than during infusion of saline. Supplementation of captopril to drinking water did not influence resting MABP and cardiovascular responses to air jet stress either in the rats receiving saline or in those subjected to V1R blockade. It is concluded that chronic blockade of central V1R significantly decreases resting MABP and reduces cardiovascular responses to alarming stress in chronically stressed infarcted rats whereas oral administraton of captopril appears to be not effective. The study was supported by grants from Ministry of Education and Science (2 PO5A 161 30) and Medical University of Warsaw (1MA/N2/2005‐07).
Resting Levels and Baroreflex Control of Cardiac and Renal Sympathetic Nerve Activity in Heart Failure
Authors: Ramchandra Rohit , Hood Sally G. , May Clive N.
Published: June 2021

During heart failure (HF), cardiac noradrenaline release is increased by up to 50‐fold, a much greater increase than from other organs. The reasons behind this preferential activation of cardiac sympathetic nerve activity (SNA) remain unclear. We hypothesised that the basal level of cardiac SNA in normal animals is set at a lower percentage of its maximum than is SNA to other organs. To investigate this possibility we measured cardiac SNA and renal SNA simultaneously in conscious sheep, at least 4 days after surgery. Experiments were conducted in normal (n = 7) and HF animals (n = 7). Consistent with our hypothesis, the burst frequency of cardiac SNA was significantly lower than renal SNA in normal animals (35 ± 7 % cardiac vs. 92 ± 2 % renal; P<0.05). Furthermore, the resting level of cardiac SNA was set at a lower percentage of maximum compared to renal SNA. HF was associated with an increase in the baseline activity of both nerves to similar levels compared to their maximum. HF was not accompanied by a change in baroreflex gain of either cardiac or renal SNA compared to normal animals. Our results indicate that in the normal state the resting level of cardiac SNA is set lower than renal SNA and during HF the SNA to both organs are increased to similar levels of maximum. This result offers one explanation for the preferential increase in cardiac noradrenaline spillover observed in HF. Funded by NHMRC, Australia and NIH.
Contribution of synaptic inhibition to the control of inspiratory hypoglossal motoneuronal (IHMN) activity in vivo
Authors: Sanchez Antonio , Mustapic Sanda , Stucke Astrid S. , Zuperku Edward J. , Stuth Eckehard E.A.
Published: June 2021

We examine the role of endogenous γ‐aminobutyric acid (GABA)ergic and glycinergic inputs on IHMN discharge activity in decerebrate, vagotomized, paralyzed, and mechanically ventilated dogs during hypercapnic hyperoxia. The GABAA receptor antagonist bicuculline (BIC) (200 μM), the GABAA receptor antagonist picrotoxin (PIC) (5 mM), or the glycine receptor antagonist strychnine (STR) (200 μM) was picoejected onto extracellularly‐recorded single IHMNs using multibarrel micropipettes at increasing dose‐rates until no further change in neuronal discharge frequency was observed. Presence of GABAA and glycine receptors and effectiveness of the antagonist doses were confirmed by picoejection of GABA (2 mM) or glycine (2mM) with subsequent block by the respective antagonists. At maximally effective BIC dose‐rates, the peak frequency of 20 IHMNs increased by 29 ± 30%. BIC also increased the slope of the control inspiratory phase discharge pattern by 25 ± 28%. Neither PIC (n=17, 5 ± 19%), nor STR (n =24, 3 ± 15%) had a significant effect on discharge activity. These data suggest that the basal activity of IHMNs is attenuated by a BIC‐sensitive tonic GABAergic inhibition, but phasic GABAergic inhibition and glycine receptors, although present, are not involved in controlling the activity of these neurons. Support: VA Medical Research Funds & NIH R01 GM59234.
Somatic and proximal dendritic Ca2+ dynamics in inspiratory preBotzinger Complex neurons.
Authors: Morgado‐Valle Consuelo , Cheng Adrian , Fredrickson Lea , Beltran‐Parrazal Luis , Arisaka Katsushi , Feldman Jack L
Published: June 2021

PreBötzinger Complex (preBötC) neurons are interconnected and postulated to underlie respiratory rhythm generation. Each inspiration results from preBötC neurons firing a synchronous burst of action potentials on top of a 10–20 mV, 0.3–1 s inspiratory drive potential. The mechanisms for inspiratory drive generation remain unknown. We tested whether somatic and proximal dendritic [Ca2+] increases during inspiratory drive. We used whole cell recording and a CMOS camera coupled to an intensifier to detect single neuron Ca2+ signals with high temporal resolution (1 kHz) in neonatal rat medullary slice preparations, recording simultaneously the integrated rhythmic hypoglossal nerve activity (∫XIIn). In neurons loaded with Fluo‐4, we were able to resolve somatic and proximal dendritic Ca2+ transients associated with each AP. We isolated the inspiratory drive potential after preventing APs by blocking Na+ channels with intracellular QX‐314. In the absence of APs, we did not detect somatic or proximal dendritic Ca2+ signals contributing to the inspiratory drive potential. Our data suggest that during inspiration somatic and proximal dendritic Ca2+ transients are mainly the consequence of Ca2+ influx through VGCCs during backpropagating APs. We propose that if Ca2+ influx contributes to inspiratory drive formation is through Ca2+ transients happening at distal dendrites by synaptic events that modulate/activate drive currents. However, we recognize the possibility that extremely low level or highly localized [Ca2+] transients, undetectable with our current technology, could be present.
Progressive Heat Storage during Intermittent Work
Authors: Kenny Glen Patrick , Dorman Lucy , Gagnon Daniel , DuCharme Michel , Webb Paul , Reardon Francis , Jay Ollie
Published: June 2021

Ten participants (5M, 5F) completed 3 × 30‐min bouts of exercise (Ex 1, 2, 3) at a metabolic heat load (M‐W) of 505±7 W separated by 3 × 15‐min recovery (Rec 1, 2, 3) in an air calorimeter at 30°C, 30% RH. Rates of evaporative heat loss (EHL), dry heat loss (DHL) and M‐W were measured throughout by whole‐body direct calorimetry, giving changes in body heat content (ΔHb). Rectal (Tre), quadriceps (Tquad) and triceps (Ttri) temperature were measured throughout. ΔHb progressively increased from the end of Ex 1 (242±18 kJ) to end Ex 2 (308±29 kJ), to end Ex 3 (341±46 kJ). Heat content dissipated during Rec 1 (−63±10 kJ) was similar to Rec 2 (−74±9 kJ) and Rec 3 (−73±7 kJ). Tre progressively increased from 36.88±0.11°C at rest to 37.41±0.12°C at the end of Ex 1, 37.64±0.12°C (Ex 2), and 37.74±0.12°C (Ex 3). Ttri increased from 33.91±0.19°C at rest, to 34.86±0.25°C (Ex 1), 35.23±0.28°C (Ex 2), and 35.40±0.33°C (Ex 3). However, after an initial increase in Tquad from 34.62±0.14°C at rest to 36.82±0.16°C (Ex 1), no further significant elevations were observed. DHL was between ~50 and 65 W throughout. At the end of Ex 1 (383±25 W), Ex 2 (398±9 W) and Ex 3 (413±9 W) EHL was similar; however EHL increased at a greater rate during Ex 2 and 3 relative to Ex 1. Progressive elevations in body heat content, core temperature and inactive muscle temperature occurred with intermittent exercise.

Supported by NSERC and US Army Medical Research Grant.
Restoration of fasting‐induced torpor in dopamine beta hydoxylase −/− mice
Authors: Swoap Stevn John , Weinshenker David
Published: June 2021

Many organisms throughout the animal kingdom, including mice, can experience a well‐orchestrated decline in metabolic rate and core body temperature (Tb) in response to, or in anticipation of, a lack of sufficient caloric intake. Previous investigations have shown that the adipocyte‐derived hormone leptin can blunt or even prevent these bouts of torpor. Unlike wildtype counterparts, mice deficient in the enzyme dopamine β hydroxylase (Dbh −/− mice), which are unable to synthesize epinephrine and norepinephrine, do not reduce circulating leptin upon fasting nor do they enter torpor. To test the hypothesis that the onset of torpor bouts in mice during a fast requires sympathetically‐mediated reduction in circulating leptin, mice deficient in both leptin and the enzyme dbh (DBL KO) were generated. Both ob/ob mice, which are deficient in leptin, and the DBL KO mice were obese at 6 months of age (approximately 60 grams) and ate 60% more food (5 grams per day vs. 3 grams per day for counterparts). This suggests that Epi and NE play little role in the mechanism of leptin‐deficient obesity. Upon fasting, wildtype and ob/ob mice entered torpor as assessed by telemetric core Tb acquisition (WT min Tb = 23.9 ± 0.4: ob/ob min Tb = 23.8 ± 0.4 C), whereas the length of torpor was significantly longer in ob/ob mice vs. WT (633 ± 53 vs. 359 ± 35 minutes, respectively). Both ob/ob and WT mice entered torpor about 7 hours after initiation of the fast. As we have seen previously Dbh −/− mice did not enter torpor with fasting. The DBL KO mice entered torpor upon fasting (min. Tb = 24.3 ± 0.4 C), did so within the first 30 minutes of fasting, and remained in the torpor bout twice as long as ob/ob mice (1155 ± 42 minutes). These data are consistent with our hypothesis that sympathetic activation of white fat is required for the onset of torpor, and that catecholamines are important in recovery from a bout of torpor.
Quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ™) reveals differential protein expression in sedentary and exercised trained cardiac mitochondria
Authors: Kavazis Andreas N , McClung Joseph M , Alvarez Sophie , Powers Scott K
Published: June 2021

Endurance exercise is known to provide cardioprotection against ischemia‐reperfusion induced myocardial injury, and mitochondrial adaptations may play a critical role in this protection. To investigate exercise‐induced changes in mitochondrial proteins, we compared the proteome of both subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria isolated from hearts of sedentary and exercise trained rats. To achieve this goal, we utilized iTRAQ™, a new method that allows identification and quantification of proteins between multiple samples. Using the Paragon™ algorithm from Protein Pilot™ software, we identified a total of 187 and 173 proteins with 95% confidence in SS and IMF mitochondria, respectively. Out of the 187 proteins identified in SS mitochondria, 25 increased in abundance and 23 decreased (Error Factor (EF) < 2 and p < 0.05) following exercise training. In IMF mitochondria, 27 proteins increased in abundance and 28 proteins decreased after exercise training (EF < 2 and p < 0.05). Importantly, expression of peroxiredoxin‐3 was significantly increased in both SS and IMF mitochondria, and the 10 kDa heat shock protein expression significantly increased in IMF mitochondria following exercise training. We conclude that exercise training induces alterations in the mitochondrial proteome that may contribute to a cardioprotective phenotype. Supported by NIH R01HL067855 (SKP).
Attenuated endothelium response to acetylcholine in abdominal aorta of Otsuka Long‐Evans Tokushima Fatty (OLETF) rats: Effects of physical activity (PA)
Authors: Bunker Aaron Kristopher , Thorne Pam , Arce‐Esquivel Arturo , Laughlin M. Harold
Published: June 2021

We tested the hypothesis that PA attenuates development of decreased endothelium‐dependent relaxation (EDR) in the aorta of OLETF rats. Rats were divided into sedentary (SED) and PA (had access to running wheels) groups at 4 weeks of age (W). Control rats were aged matched Long‐Evans Tokushima Otsuka (LETO). At 13W and 20W acetylcholine (Ach) was used to examine EDR. To evaluate the role of nitric oxide (NO) and cyclooxygenase (COX), EDR was also evaluated following treatment with N‐nitro‐L‐arginine methyl ester (L‐N) and/or Indomethacin (I). EDR was similar in 13W‐OLETF‐SED & ‐PA aortas. L‐N+I treatments similarly blocked Ach EDR in all 13W groups, except for a 30–40% Ach‐induced EDR in LETO‐SED, suggesting a role for endothelium dependent hyperpolarizing factor (EDHF) in EDR. At 20W OLETF‐SED maximal Ach EDR was less than in 13W OLETF‐SED. EDR was not decreased in 20W OLETF‐PA. Ach‐induced EDR was about 30% in all 20W groups, after treatment with L‐N+I. Results indicate an increased role for EDHF in EDR in 20W OLTEF aortas, and blunted EDR in OLETF‐SED rats at 20W, but not in 20W OLETF‐PA rats. The 30% Ach EDR in L‐N+I treated 20W OLETF aortas, not seen in 20W L‐N, 13W L‐N, or 13W L‐N+I treated aortas, indicates a possible increase of COX constrictor pathways during development of blunted EDR. Support: HL‐36088 and HL‐29420
Exogenous ATP abolishes postjunctional α1‐ and α2‐adrenergic vasoconstriction in humans
Authors: Kirby Brett S , Carlson Rick E , Voyles Wyatt F , Dinenno Frank A
Published: June 2021

Recent evidence suggests that adenosine triphosphate (ATP) can inhibit vasoconstrictor responses to endogenous norepinephrine release via tyramine, similar to what is observed in contracting muscle. Whether this involves a reduction in postjunctional α‐adrenergic receptor responsiveness is unclear. Therefore, we tested the hypothesis that exogenous ATP can blunt direct postjunctional α‐adrenergic vasoconstriction in humans. We measured forearm blood flow (FBF; Doppler ultrasound) and calculated the vascular conductance (FVC) responses to local intra‐arterial infusions of phenylephrine (α1‐agonist) and dexmedetomidine (α2‐agonist) during moderate rhythmic handgrip exercise, during a control non‐exercise vasodilator condition (adenosine), and during ATP infusion in 7 young adults. Forearm hyperemia was matched across all conditions. Forearm vasoconstrictor responses to direct α1‐receptor stimulation were blunted during exercise vs adenosine (ΔFVC = −11±3% vs −36±5%; P<0.05), and were abolished during ATP infusion (−4±3%). Similarly, vasoconstrictor responses to α2‐receptor stimulation were blunted during exercise vs adenosine (−12±4% vs−37±9%; P<0.05), and were abolished during ATP infusion (−3±5%). We conclude that exogenous ATP abolishes postjunctional α‐adrenergic vasoconstriction, and this involves both α1‐ and α2‐receptor subtypes.

Supported by AG‐022337, HL‐087952
Local H1‐and H2‐receptor blockade blunts postexercise blood flow in the vastus lateralis
Authors: Pellinger Thomas K , Simmons Grant H , Halliwill John R
Published: June 2021

Orally administered histamine H1‐ and H2‐receptor blockade blunts the sustained (90 min) hyperemia present in the previously exercised limbs of humans following dynamic exercise. Our aim was to determine if this postexercise hyperemic response could be prevented by local H1‐ and H2‐receptor blockade administered via skeletal muscle microdialysis. Thus, we tested the hypothesis that postexercise ethanol outflow/inflow ratios (an inverse surrogate of local blood flow) would be higher in sites that received H1‐ and H2‐receptor blockade versus control sites. Four microdialysis probes were inserted into the vastus lateralis muscle of five healthy subjects (21–26 yrs) immediately after 60 min of upright cycling at 60% VO2 peak. Microdialysis probes were perfused with a modified Ringer's solution containing 5 mM ethanol. Two of the sites received H1‐ and H2‐receptor antagonists (1 mM pyrilamine/3 mM cimetidine) and two sites served as controls. Consistent with our hypothesis, one hour after exercise, the outflow/inflow ratio was 0.48 ± 0.02 in blockade sites versus 0.41 ± 0.03 in control sites (P = 0.042). In addition, three subjects underwent a sham protocol (no exercise) in which blockade did not affect the ethanol outflow/inflow ratio (P ≥ 0.143). These results suggest that local H1‐ and H2‐receptors in skeletal muscle contribute to postexercise hyperemia.

Supported by AHA grant 555623Z and the Evonuk Foundation
Does acute dynamic exercise improve brain cognitive function in humans?
Authors: Endo Kana , Tsuchimochi Hirotsugu , Nakamoto Tomoko , Kashima Eri , Matsukawa Kanji , Okamura Hitoshi , Hamaoka Takafumi
Published: June 2021

To test the hypotheses that 1) acute dynamic exercise can improve cognitive function and 2) changes in cerebral blood flow of the frontal brain may be associated with improved cognitive function, a Stroop color‐word test (SCWT) was performed in 12 subjects before and 5 min after cycling exercise for 15 min at 20%, 40%, and 60% of maximum voluntary exercise. SCWT can evaluate quantitatively cognitive function. When a “color word” was displayed in a color different from “the color's meaning”, the subjects were asked to quickly answer the color of the word. The total time and the number of errors in 100 SCWT trials were recorded as index of cognitive function. We measured the concentration of oxy‐hemoglobin (O2Hb) in the frontal brain with a near‐infrared spectroscopy to monitor local cerebral blood flow. The total time for SCWT was shortened and O2Hb was increased only by 40%‐ and 60%‐exercise. The increase in O2Hb started 5 min after the onset of the exercise. The number of error was not altered by any intensity of exercise. When the duration of 40%‐exercise was shortened to 5 min in 6 subjects, both the total time for SCWT and O2Hb were not altered. These results suggest that 1) acute dynamic exercise at a moderate intensity for 15 min, but not 5 min, is able to improve brain cognitive function and 2) the improved cognitive function may be in association with increased cerebral blood flow of the frontal brain.
Palmitic Acid Treatment Decreases C2C12 Myoblasts Proliferation Rates through a G2 cell cycle shift
Authors: Peterson Jonathan M , Bryner Randy W , Alway Stephen E
Published: June 2021

Metabolites of palmitate, i.e. ceramide, may have a number of detrimental effects. The purpose of this study was to determine if palmitic acid would have a negative effect on myoblast proliferation and Akt activation. Myoblasts were grown treated with 0.50 mM palmitate in 2% BSA for 12 hours; Laurate and BSA only were used as controls. Proliferation was determined by exposing the myoblasts to CFSE; CFSE fluorescent intensity is reduced in half each time the cell divides. Immunoblot analysis revealed that palmitate resulted in a ~50% reduction in Akt protein expression and ~65% reduction in the amount of Akt phosphorylated at ser473 compared to both BSA and laurate treated controls. In addition, FACS analysis of CSFE intensity demonstrated that palmitate treatment decreased myoblast proliferation by 33 + 6% compared to BSA only control and 24 + 2% compared to laurate treated myoblasts (p < 0.01). Furthermore, FACS analysis of propidium iodide staining demonstrated that there was greater than two‐fold increase in the percent of cells in the G2 phase of cell cycle with palmitate treatment compared to either of the control treatments. These data demonstrate that palmitate treatment reduced Akt activation, as indicated by decreased serine 473 phosphorylation, reduced myoblast proliferation, and caused a G2 shift in the cell cycle progression.
mTOR signaling following Essential Amino Acid + Carbohydrate Ingestion in Young and Older Subjects
Authors: Dreyer Hans Christian , Fujita Satoshi , Drummond Micah John , Rasmussen Blake , Volip Elena
Published: June 2021

Essential amino acid (EAA) ingestion can stimulate muscle protein synthesis in young and older subjects. In older subjects however, the addition of carbohydrate (CHO) (EAA+CHO) has been shown to blunt the anabolic response as compared to young subjects and may be due to dysregulation of the anabolic (mTOR) signaling pathway.

PURPOSE:

The purpose of this study was to determine the signaling response after ingestion of EAA + CHO in young and older muscle and to determine if the response between groups was different.

METHODS:

Immunoblotting methods were used to determine cell signaling of an anabolic pathway (mTOR), at 1 and 2hr post EAA+CHO ingestion.

RESULTS:

Our preliminary results from 4 young and 5 older subjects demonstrate that Akt, mTOR, S6K1 and 4E‐BP1 phosphorylation increased following EAA+CHO ingestion while eEF2 phosphorylation decreased only in young. In addition there was a trend for 4E‐BP1 phosphorylation to be greater in the young at 1h post ingestion (P=0.09).

CONCLUSIONS:

Our preliminary findings suggest that a potential differential response to EAA+CHO ingestion exists between young and old. This response may indicate that both translation initiation (4E‐BP1 phosphorylation) and elongation (eEF2 dephosphorylation) may be attenuated in the old. Further research in needed to elucidate the potential differential response between young and old muscle following EAA+CHO ingestion.

This work was supported by a Pepper Pilot Project award
Investigating the mechanism of regulation of muscle glycogen synthase
Authors: Sakamoto Kei , Bouskila Michale , Ibrahim Adel , Peggie Mark , Hirshman Michael F , Goodyear Laurie J , Jensen Jorgen
Published: June 2021

Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating GSK3. Insulin also promotes glucose uptake and glucose‐6‐phosphate (G6P) production, which allosterically activates GS. The aims of this study were (1) to determine if the inhibition of GSK3 is necessary for insulin‐mediated muscle glycogen synthesis in vivo and (2) to identify the molecular mechanism by which G6P regulates GS. We used knock‐in (KI) mice in which GSK3α and β genes are replaced with constitutively‐active forms (GSK3α/βS21A/S21A/S9A/S9A). Insulin‐induced dephosphorylation and activation of GS was abolished in GSK3 KI mice, however muscle glycogen content from these mice was not reduced. Injection of [3H]2‐deoxyglucose and [14C]glucose resulted in similar rates of muscle glucose uptake and glycogen synthesis in vivo between WT and GSK3 KI. These results provided genetic evidence that inhibition of GSK3 and subsequent dephosphorylation of GS are not essential for muscle glycogen synthesis in vivo, indicating that allosteric regulation of GS by G6P may play a key role in this process. We next mutated putative G6P binding residues of GS and expressed these mutants in HEK293 cells and activity assessed. We identified that Arg582/586 residues are responsible for G6P‐mediated activation of muscle GS. Characterization of these mutants will allow us to determine the role that G6P regulates GS and glycogen levels in vivo.
Antagonism of endocannabinoid receptor CB1 directly increases basal and insulin‐stimulated glucose transport activity in soleus muscle of lean Zucker rats
Authors: Lindborg Katherine Ann , Teachey Mary K. , Henriksen Erik J.
Published: June 2021

The endocannabinoid system is emerging as a new pharmacological target in the treatment of obesity and insulin resistance. It is known that endocannabinoid receptors (CB1 subtype) are expressed in rat skeletal muscle. However, it is currently unknown what role, if any, modulation of these receptors might have on the glucose transport system in skeletal muscle. Therefore, the goal of this study was to determine whether glucose transport activity in soleus muscle of lean Zucker rats is directly affected by selective CB1 receptor inhibition. Soleus muscle strips from lean Zucker rats were incubated for 60 min in the absence or presence of a maximally‐effective insulin concentration (5 mU/ml) and increasing concentrations (25–1000 nM) of the CB1 receptor antagonist, SR 141716. In the absence of insulin, SR141716 at 100–200 nM activated glucose transport (2‐deoxyglucose uptake) by 33% (p<0.05), with an EC50 of ~50 nM. Insulin‐stimulated glucose transport was enhanced 37% (p<0.05) by 100 nM SR141716, again with an EC50 of ~50 nM. These SR141716‐mediated increases were substantially reduced at the highest concentrations of the CB1 receptor antagonist. These results support the hypothesis that the glucose transport system in rat skeletal muscle is directly and positively modulated by CB1 receptor antagonism.
Serum starvation increases phosphorylation of the AMP‐activated protein kinase (AMPK) in myotubes
Authors: Ching James Kain , Rajguru Pooja , Marupudi Nandhini , Banerjee Sankha , Fisher Jonathan S.
Published: June 2021

The signaling events that link serum starvation to increased insulin action in myotubes have not been defined. We hypothesized that serum starvation enhances phosphorylation of the AMP‐activated protein kinase (AMPK) and other possible mediators of insulin sensitivity, including Akt, p38, protein kinase C zeta (PKCζ), and the Akt substrate of 160 kDa (AS160). Differentiated L6 myotubes were incubated in serum‐containing or serum‐free medium for three hours. AMPK and Akt S473 phosphorylation were ∼40% and ∼80% greater, respectively, in serum‐starved cells than in control cells (P<0.05). Serum starvation did not affect p38, PKCζ, or AS160 phosphorylation or insulin‐stimulated Akt phosphorylation. Insulin had no effect on glucose transport in control cells but caused a ∼40% increase in glucose uptake for serum‐starved cells (P<0.05). This insulin action in serum‐starved cells was prevented by Compound C, an AMPK inhibitor. Thus, it appears that potentiation of insulin action by serum starvation is mediated by an increase in AMPK phosphorylation. These data suggest that the potential for increased AMPK phosphorylation should be a consideration in experimental settings requiring serum starvation.
Identification and characterization of a novel Muscle Lamin A/C (LMNA) Interacting Protein (MLIP): A regulator of Pax7
Authors: Ahmady Elmira , Burgon Patrick G
Published: June 2021

Specific missense mutations LMNA have been identified to be associated with muscular dystrophy suggesting that LMNA interacts with a muscle specific factor(s). A yeast two‐hybrid screen with LMNA as bait was employed to identify muscle specific proteins. A previously uncharacterized cDNA clone was identified that interacts with LMNA in muscle, MLIP. Preliminary results show MLIP protein is primarily expressed in brain, heart and skeletal muscle. No structural or functional domains have been identified within MLIP.

Objective: To define the function of MLIP in muscle.

Results: MLIP is expressed endogenously in a mouse myoblast cell line (C2C12) and is co‐localized to the nuclear envelope and PML bodies. Initiation of C2C12 differentiation led to a 3‐fold increase in MLIP expression peaking at 24hrs and MLIP continued to be expressed in differentiated myotubes. Specific knockdown of MLIP in C2C12 cells by shRNAi led to significant (p<0.01) reduction in Pax7 expression with a concurrent reduction in Myf5, MyoD and myogenin with Pax3 expression being unaffected. To determine whether MLIP is necessary and/or sufficient for myogenic differentiation, stable C2C12 cell lines are being generated that either knockdown MLIP or over‐express MLIP.

Conclusion: MLIP is a novel LMNA interacting protein that may regulate Pax7 a myogenic determinant during regenerative myogenesis. Funding: CIHR operating grant to PGB.
REDD2 inhibits mTOR signaling in skeletal muscle cells
Authors: Miyazaki Mitsunori , McCarthy John J , Zhang Xiping , Esser Karyn A
Published: June 2021

The protein kinase mTOR (mammalian target of rapamycin) is established in skeletal muscle as a key signaling hub during cell growth/hypertrophy. In this study, we determined that the stress responsive gene REDD2 inhibits mTOR signaling in skeletal muscle cells. The effects of REDD2 expression on mTOR activity were also determined in C2C12 muscle cell. Overexpression of REDD2 resulted in decreased mTOR kinase activity under both basal and amino acid‐stimulated conditions. These inhibitory effects on mTOR signaling are mediated downstream of Akt but upstream of Rheb. Knock down of TSC2 (tuberous sclerosis complex 2) using siRNA potently activated mTOR signaling and was sufficient to rescue REDD2 inhibition of mTOR activity. Consistent with the in vitro studies, REDD2 mRNA levels are rapidly reduced in the plantaris muscle during hypertrophy and increased in vivo during atrophic conditions. These results indicate that REDD2 negatively regulates mTOR activity likely through modulation of TSC2 function and regulating of REDD2 might regulation of muscle cell size in mice.

This study is supported by the grant provided from National Institute of Health (AR45617).