bluuebunny/crossref_metadata_2025 huggingface dataset:
The SIRT3 deacetylase blocks cardiac fibrosis by targeting TGFβ1‐Smad3 signaling (1152.6)
Authors: Gupta Mahesh , Sundaresan Nagalingam , Gupta Madhu
Published: June 2021

Cardiac fibrosis after myocardial injury is a major challenge in the medicine today to preserve heart function. At the cellular level, activation of TGFβ1 signaling is considered a major contributor of cardiac fibrosis. However, the underlying mechanism of TGFβ activation is not yet completely understood. SIRT3 is a mitochondrial deacetylase which is activated by exercise and calorie restriction. SIRT3 regulates variety of mitochondrial functions, including metabolism, ROS synthesis and apoptosis. SIRT3KO mice spontaneously develop cardiac fibrosis with age. This study was undertaken to test the hypothesis whether SIRT3 deficiency leads to transformation of cardiac fibroblasts (CF) into myofibroblasts (myoFB) and excessive synthesis of extracellular matrix (ECM). CF obtained from adult rat hearts were subjected to SIRT3 knockdown (KD) by using siRNA. Differentiation of CF to myoFB was determined by increased expression of myoFB markers, like smooth muscle α‐actin, fibronectin and collagen1. The results showed robust expression of myoFB markers in SIRT3KD cells. To test whether SIRT3 deficiency contributed to this phenotype, we re‐expressed SIRT3 and found that this intervention changed back myoFB phenotype of SIRT3‐deficient cells to normal CF. Similar results were obtained when TGFβ1‐induced myoFB were over expressed with SIRT3, thus demonstrating the ability of SIRT3 to block transformation of CF into myoFB and synthesis of excessive ECM. Mechanistic studies showed increased levels of TGFβ1 and Smad3 in SIRT3KD cells, which were resistant to use of the anti‐oxidant, N‐acetyl cysteine. These data thus demonstrated an anti‐fibrotic role of SIRT3, which is independent of its ability to regulate cellular oxidative stress.

Grant Funding Source: NIH‐NHLBI
Vascular endothelial growth factor‐C: its unrevealed role in fibrogenesis (1152.9)
Authors: Zhao Tieqiang , Zhao Wenyuan , Meng Weixin , Liu Chang , Chen Yuanjian , Sun Yao
Published: June 2021

Vascular endothelial growth factor (VEGF)‐C is a key mediator of lymphangiogenesis. Our recent study shows that VEGF‐C/VEGF receptors (VEGFR)‐3 are significantly increased in the infarcted rat myocardium, where VEGFR‐3 is expressed not only in lymph ducts, but also in myofibroblasts, indicating that VEGF‐C has an unrevealed role in fibrogenesis during cardiac repair. The current study is to explore the regulation and molecular mechanisms of VEGF‐C in fibrogenesis. The potential regulation of VEGF‐C on myofibroblast differentiation/growth/migration, collagen degradation/synthesis and TGF‐β/ERK pathways was detected in cultured cardiac myofibroblasts. Our results showed that VEGF‐C significantly increased myofibroblast proliferation, migration and type I/III collagen production. Matrix metalloproteinase (MMP)‐2 and ‐9 were significantly elevated in the medium of VEGF‐C treated cells, coincident with increased tissue inhibitor of metalloproteinase (TIMP)‐1 and TIMP‐2. Furthermore, VEGF‐C significantly elevated TGF‐β1 synthesis and ERK phosphorylation in myofibroblasts, which was significantly suppressed by TGF‐β or ERK blockade. This study indicates that in addition to lymphangiogenesis, VEGF‐C is also involved in fibrogenesis in the repairing heart through stimulation of myofibroblast proliferation, migration and collagen synthesis, via activation of the TGF‐β1 and ERK pathways.

Grant Funding Source: NIH
Mitochondrial superoxide mediates TNF‐α‐induced myokine release from C2C12 myotubes (1153.4)
Authors: Lightfoot Adam , Sakellariou Giorgos , Nye Gareth , McArdle Francis , Jackson Malcolm , Griffiths Richard , McArdle Anne
Published: June 2021

TNF‐α is a key inflammatory mediator and is proposed to induce transcriptional responses via the generation of Reactive Oxygen Species (ROS). The aim of this study was to determine the role of mitochondrial superoxide generation in TNF‐α ‐ induced generation of myokines by skeletal muscle. Significant elevation in the release of IL‐6 (control untreated: 20.1pg/ml ± 2.3; TNF‐α treated: 73.8pg/ml ± 9.3), CCL2/MCP‐1 (980pg/ml ± 74.8; 31194pg/ml ± 5409), CCL5/RANTES (22.4pg/ml ± 2.09; 604pg/ml ± 93.4) and CXCL1/KC (236pg/ml ± 32.6; 3477pg/ml ± 383) and in superoxide, localised to the mitochondria were seen in response to TNF‐α treatment of muscle cells and this was associated with activation of NFκB. The TNF‐α mediated changes in superoxide, activation of NFκB and increased release of myokines were attenuated following pre‐treatment with the mitochondria targeted antioxidant, SS‐31 peptide (IL‐6, 11.9pg/ml ± 1.83; MCP‐1, 11322pg/ml ± 1124; RANTES, 252pg/ml ± 51.4; KC, 1166pg/ml ± 204) indicating that the ability of TNF‐α to induce myokine release is mediated through mitochondrial superoxide, which is, at least in part, associated with activation of the redox sensitive transcription factor NFκB. These novel findings provide evidence that targeting mitochondrial ROS could provide a potential for therapy for inflammatory myopathies. The authors thank the Medical Research Council and National Institute on Aging (grant AG‐020591) for their generous financial support and Dr M.B Reid (University of Kentucky Center for Muscle Biology) for the kind gift of the SS‐31 peptide.
Musculoskeletal changes in superoxide dismutase 1 null mice (1153.5)
Authors: Nye Gareth , Lightfoot Adam , Sakellariou George , Boudiffa Maya , Bellantuono Illaria , Brooks Susan , Richardson Arlan , Jackson Malcolm , Van Remmen Holly , Jeffery Nathan , McArdle Anne
Published: June 2021

SOD1 null (SOD1‐/‐) mice are a powerful model to study the effects of ageing on musculoskeletal tissues. The aim of this study was to determine the effect of lack of SOD1 in mice on mechanical changes in bones compared with old mice and to determine whether changes are due to a direct effect of the deletion in bone or indirectly muscle changes. The study used adult and old wild‐type (WT), adult SOD1‐/‐ and SOD1‐/‐ mice with expression of SOD1 rescued in nerve (SynTgSod1‐/‐) or specifically deleted in the muscle (mKO). Muscles force generation was measured and MicroCT analysis performed on tibia. Finite Element (FE) models were developed to map strain across the tibia and these data were compared with MicroCT data. Old WT mice showed a 20+/‐3% loss of muscle mass, thinning of cortical bone and a 37+/‐1% loss of trabecular bone compared with adult mice. Loss of muscle in adult SOD1‐/‐ mice was associated with a 29+/‐1% loss of trabecular bone. FE analyses indicated that this loss of muscle led to 50+/‐ 0% reduction in tibia strain which was mirrored by microCT data. SynTgSod1‐/‐ mice preserved muscle mass but showed loss of tibia trabecular bone. Muscles of mKO mice had a reduced specific force but tibia displayed normal structure. Data suggest that loss of trabecular bone in SOD1‐/‐ mice is due to the direct effect of lack of SOD1 in the bone. Funded by MRC‐ARUK Centre for Integrated research into Musculoskeletal Ageing and NIA (AG‐20591).
Chronic hyperinsulinemia impairs insulin signaling and downregulates uncoupling protein 3 in mouse heart (1155.4)
Authors: Harmancey Romain , Haight Derek , Taegtmeyer Heinrich
Published: June 2021

Chronic hyperinsulinemia is a consequence of insulin resistance and one of the main features of type 2 diabetes. To investigate the effect of hyperinsulinemia on myocardial insulin signaling and gene expression, C57BL/6 mice were treated twice daily for 15 days with gradually increasing concentrations of intermediate‐acting insulin (0.14 to 1.68 U Novolin N per day). Diet was supplemented with sucrose cubes and 5% glucose in the drinking water to prevent hypoglycemia. While chronic insulin treatment raised plasma insulin levels from 0.88 to 2.07 ng/ml (P < 0.01), it decreased Akt activity by 39% (P < 0.05) when compared to untreated mice. This was accompanied by a dramatic decrease in mitochondrial uncoupling protein 3 (UCP3) mRNA (‐51%; P < 0.001) and protein levels (‐28%; P < 0.05). Conversely, acute stimulation of the Akt pathway with a single injection of short‐acting Humulin R (1 U/kg body weight) in overnight‐fasted mice induced a 3‐fold increase in myocardial UCP3 mRNA levels. We conclude that chronic hyperinsulinemia impairs insulin signaling in the heart, which, in turn, impairs UCP3 expression. The decrease in UCP3 may link insulin resistance to mitochondrial dysfunction in the heart.

Grant Funding Source: Supported by K99 HL112952 (to R.H.) and R01 HL061483 (to H.T.)
Hemodynamic effects of β1‐ and β2‐adrenoceptor stimulation in conscious Zucker diabetic fatty rats (1155.7)
Authors: Cook Rosalind , Bussey Carol , Cragg Patricia , Lamberts Regis
Published: June 2021

The β‐adrenoceptor (β‐AR) functional responsiveness and β1‐AR expression, key components of blood pressure regulation, are reduced in many cardiomyopathies. Chronic heart failure in vitro models suggests that β2‐AR redistribution might compensate for the decreased β1‐AR function. Whether similar β‐AR subtype changes contribute to cardiovascular dysfunction in diabetes is unknown. Therefore, we aimed to determine the hemodynamic effects of specific β1‐ and β2‐AR stimulation in type II diabetes in vivo.

Male Zucker Diabetic Fatty (ZDF) rats and paired lean littermates (20 week old n=8 per group) were implanted with a radiotelemeter and vascular access port to measure arterial blood pressure, derive heart rate (HR) and inject drugs intravenously under stress‐free conscious conditions. Cumulative doses of isoproterenol (non‐selective β1‐ and β2‐AR agonist, 0.001‐3µg/kg) or fenoterol (non‐selective β2‐AR agonist, 0.001‐3µg/kg) in the presence of atenolol (selective β1‐AR antagonist, 2000µg/kg) were injected.

The increase in HR due to non‐selective β‐AR stimulation was not significantly different between ZDF and lean rats for all doses (at 0.1µg/kg: 106±10 vs. 109±13 bpm). However the HR response to specific β2‐AR stimulation was significantly reduced in ZDF rats (25±4 vs. 2±4 bpm and 47±7 vs. 20±2 bpm; p<0.002 at 0.03 and 0.1µg/kg, respectively).

Thus, in conscious diabetic ZDF rats the functional β‐AR responsiveness was not affected, whereas the sensitivity to specific β2‐AR stimulation was reduced. Therefore, hemodynamic regulation by β1‐ and β2‐AR is altered in diabetes, and furthermore β1‐AR signalling might play a compensatory role in maintaining β‐AR reactivity.
Reliability of finger blood pressures with simultaneous ipsilateral radial applanation tonometry (1156.11)
Authors: Harwood Morton , Strong Chelsea , De Cramer Courtney , Carter Jason , Durocher John
Published: June 2021

Important measurements in the field of autonomic physiology include assessment of continuous blood pressure via finger photoplethysmography (Finometer) and estimates of central blood pressure and arterial stiffness with applanation tonometry (SphygmoCor). It is currently unclear if finger blood pressures are influenced by radial tonometry measurements in the same limb. Reliable simultaneous measurements from the same limb would be advantageous in study designs that might require an additional measurement on the opposite arm (ex. forearm plethysmography) or intervention (ex. cold‐pressor test). We hypothesized that baseline finger blood pressures would be similar with and without simultaneous applanation tonometry. Eleven healthy participants completed a 10 min resting supine baseline, with the first 5 min free of tonometry measurements and the second 5 min with at least 30 sec of tonometry performed within each minute. Systolic arterial pressure (SAP), diastolic arterial pressure (DAP), mean arterial pressure (MAP), and heart rate (HR) were assessed during the entire 10 min baseline. SAP (118±3 vs. 119±4 mmHg), DAP (61±2 vs. 61±2 mmHg), MAP (80±2 vs. 80±2 mmHg), and HR (61±1 vs. 61±1 beats/min) were all similar (p>0.25) in the first 5 and second 5 min of baseline, respectively. We conclude that supine baseline finger blood pressures are reliable with concurrent ipsilateral radial applanation tonometry.
Increases in rat adipose blood flows following bolus intraperitoneal injection of glucose (1156.2)
Authors: Yang Hsiaotung , Rector Scott , Thorne Pamela , Chen Xinyue , Laughlin Harold
Published: June 2021

The purpose of this study was to determine the magnitude of increased blood flow (BF) to rat adipose tissue depots that is signaled by hyperglycemia. BF was measured in adult SD rats (422±2.6 g, n=6) after 5 hr fasting with stable isotope labeled microshereres. > 4 hr recovery from arterial catheterizing surgery, BF was determined in conscious rats confined to cage activity at before and 15 min after bolus dextrose (2 g/kg b.w.) intraperitoneal injection (i.p.). Blood glucose level was increased from 144±10.1 to 463±36.3 mg/dl (p<0.001). Arterial blood pressure (BP) (141±5.8 mm Hg), and heart rate (460±10.0 bpm) remained unchanged by hyperglycemia. BFs (ml/min/100 g) in adipose tissues prior to glucose i.p. were as follows: epididymal (22±2.4), retroperitoneal (200±36.4), omental (47±5.4), subcutaneous (38±2.9), and brown fat (369±66.7). Fifteen min post i.p. glucose, BFs showed 11.3, 5.3, 13.1, 3.8, and 1.4 fold increase in those adipose tissues respectively (p<0.01). In addition, hyperglycemia also induced 4‐6 fold increases in BFs in the hind limb muscles, spleen, kidney, and duodenum (p<0.01), BF to the liver was 13.0 fold higher (p<0.001) in the post compared to prior glucose i.p. (22±2.6 ml/min/100 g). The independent effects of insulin were not assessed, but could have contributed to observed changes in BF. These results demonstrate the dramatic effects of 3.2 fold elevated circulating glucose on increases in BFs in adipose, skeletal muscle, and some visceral organs.

Supported by NIH HL 36088 (MHL) , VA‐CDA2 (1299‐02) (RSR), and CVM Research Grant of MU (MHL and HTY)

Grant Funding Source: NIH HL 36088 (MHL), VA‐CDA2 (1299‐02) (RSR), and CVM Research Grant of MU (MHL and HTY)
Brain endocannabinoids modulate lung Inflammation during sepsis (1157.4)
Authors: Peng Jianya , Russo Vincenzo , Gertzberg Nancy , Neumann Paul , Johnson Arnold , Feleder Carlos
Published: June 2021

Sepsis is a systemic inflammatory response caused by infection which can lead to shock and lung injury. Previous studies indicate that septic shock is mediated by brain endocannabinoid 1 (CB1) receptors. Hence, this study tested the hypothesis that the brain CB1 receptors regulate LPS‐induced lung inflammation. Male Sprague‐Dawley rats received an intracerebroventricular (ICV) injection of either the CB1 receptor antagonist Rimonabant (2 µg/kg) or vehicle, 5 minutes prior to IV injection of either LPS (5 mg/kg) or saline. Lungs were removed 0.5 h or 4 h after IV injection of LPS and isolated for assessment of pulmonary capillary pressure (Ppc), lung wet/dry weight ratio (W/D), TLR4 activation, α7nicotinic acetylcholine (α7nAch) pathway, IL‐6 and VCAM.

LPS‐ 0.5 h induced a decrease in lung levels of IRAK1 and IκBα indicating activation of TLR4, and decreased phosphorylated Src‐Y416 family kinases (pSFK) and phosphorylated Raf1‐S259 (pRaf1) suggesting decreased α7nAch signaling all which were prevented by Rimonabant. In addition, Rimonabant prevented the LPS‐induced 4 h increase in the W/D/Ppc and IL‐6 and Rimonabant reduced the LPS induced increase in VCAM expression. The data indicates that the brain CB1 receptor mediates, in part, the lung inflammation response to LPS.

Grant Funding Source: Supported by NIH R01 HL059901 to A. Johnson and NIH R15A1072744 to C. Feleder
Impaired lung mitochondrial oxidative capacity following perinatal nicotine exposure in rats (1159.2)
Authors: Cannon Daniel , Liu Jie , Sakurai Reiko , Rossiter Harry , Rehan Virender
Published: June 2021

Perinatal smoke and nicotine exposure predisposes to chronic lung disease and morbidity. As the PPARγ pathway is implicated in structural and functional airway deficits in a well‐established rat model of perinatal nicotine exposure, mitochondrial derangement may accompany these deficits. Therefore, we hypothesized that perinatal nicotine exposure would result in lung mitochondrial dysfunction.

Sprague‐Dawley dams received either placebo (CON) or nicotine (NIC, 1 mg.kg‐1) daily from embryonic day 6 to postnatal day 21. Postpartum, pups were breast feed ad libitum. Parenchymal lung sections (~10mg) were taken from adult males for mitochondrial assessment in situ (O2K, Oroboros).

Body mass was not different at 5 months between CON and NIC (637±65 vs. 624±49 g; p=0.71). ADP‐stimulated O2 consumption (JO2) was ~50% less in NIC (n=6) compared to CON (n=6) (4.5±0.8 vs.8.8±2.5 pmol.s.mg‐1, respectively; p<0.05). Uncoupled JO2 was ~60% less in NIC (6.4±1.5 vs. 15.4±5.7 pmol.s.mg‐1; p<0.05), each with excess complex IV capacity. Flux control ratios were not different between conditions (p>0.05).

These data suggest reduced mitochondrial density, rather than respiratory chain complex dysfunction, with perinatal nicotine exposure. Low maximal JO2 with nicotine exposure, possibly via suppressed mitochondrial biogenesis, may predispose towards increased oxidative stress and chronic lung disease.

Grant Funding Source: Supported by NIH HD51857, HD71731
Systemic factors associated with adipose tissue remodeling after severe burn injury in children (1160.2)
Authors: Saraf Manish , Porter Craig , Chao Tony , Finnerty Celesete , Chondronikola Maria , Borsheim Elisabet , Herndon David , Sidossis Labros
Published: June 2021

The chronic adrenergic stress response to severe burn injury a causes plethora of metabolic abnormalities. However, the impact of burn injury on adipose tissue (AT) metabolism remains largely unknown. Here, we studied the effect of severe burn injury on systemic biomarkers thought to be involved in AT remodeling (i.e. White‐to‐brown transdifferentiation).

Ten severely burned children (age 12±4 years; % total burn surface area 49±20) and ten healthy children (age 12±5 years) participated in this study. Hormones levels were measured by luminex multiplex magnetic assay kit and ELISA kit in plasma samples obtained in the fasted state (2‐4 weeks post injury in burned patients).

Plasma fibroblast growth factor 21 (634 vs 40 pg/ml, (p=0.07), leptin (9 vs 1.2 ng/ml; p=0.055), interleukin ‐ 6 (68 vs 4.8 pg/ml; p=0.03) and amylin (65 vs 24 ng/ml, p=0.06) were all increased systemically in burn patients vs. healthy subjects. Irisin also showed a trend to be increased in burn patients (116 vs 83 ng/ml, p=0.24), however this was not statistically different. These findings suggest that severe chronic inflammatory stress alters the systemic hormonal profile of burn patients in a similar manner to that observed during adrenergic stimulation in rodents that has been shown to result in white‐to‐brown transdifferentiation.

Grant Funding Source: NIH (P50‐GM60338, R01‐GM05668) and SHC (84090, 84080, 71008 and 71006) g
Browning of subcutaneous white adipose tissue in humans after severe adrenergic stress (1160.5)
Authors: Sidossis Labros , Porter Craig , Saraf Manish , Chao Tony , Finnerty Celeste , Chondronikola Maria , Borsheim Elisabet , Hawkins Hal , Herndon David
Published: June 2021

Intriguing new evidence from animal models suggests that white adipose tissue (WAT) can acquire a brown adipose tissue (BAT) phenotype after adrenergic stress. In this study, we investigated whether the WAT of humans has the capacity to adopt a BAT‐like phenotype (browning of WAT). Subcutaneous WAT samples were collected from 20 severely burned children (early group [8±4 days post injury] or late group [37±25 days]) and 6 healthy children (control group). Morphologic assessment of WAT from the late burn group showed numerous multilocular adipocytes that stained positive for uncoupling protein 1 (UCP1). UCP1 protein content was significantly higher in the late burn group compared to healthy controls (2.72±0.31 vs. 22.88±4.45 ng/mg, P<0.001) and the early burn group (6.88±1.39 vs. 22.88±4.45 ng/mg, P<0.01). Likewise, coupled and uncoupled mitochondrial respiration in the late burn group were 3‐fold and 4‐fold greater, respectively compared to the control group (P<0.01) and the maximum activity of citrate synthase (a proxy for mitochondrial content) of the late group was 3.7‐fold (P<0.001) higher than that in the control group. These findings sugges that the observed changes in WAT are functionally significant. To the best of our knowledge, this is the first evidence of the browning of subcutaneous WAT in humans. Our data highlight the role of WAT as a potential regulator of energy balance and metabolic control in humans.

Grant Funding Source: Supported by NIH P50‐GM60338, R01‐GM05668, and Shriners Hospitals for Children (84090, 84080)
Whey protein, but not leucine ingestion reduces insulin sensitivity (1161.5)
Authors: Smith Gordon , Magkos Faidon , Klein Seth , Reeds Dominic , Bradley David , Okunade Adewole , Patterson Bruce , Klein Samuel , Mittendorfer Bettina
Published: June 2021

Intravenous infusion of an amino acid mixture reduces insulin‐mediated glucose disposal during a hyperinsulinemic‐euglycemic clamp. The mechanisms responsible for the adverse effect of amino acids on insulin‐mediated glucose disposal are unclear and it is not known whether protein ingestion has similar adverse effects. We hypothesized that protein ingestion impairs insulin‐mediated glucose disposal due to leucine‐mediated activation of mTOR and serine‐phosphorylation of IRS1. To test this hypothesis, we conducted hyperinsulinemic‐euglycemic clamps (50 mU insulin/m2 body surface area/min) in two groups (n = 9/group) of women (age: 58±1 yr, BMI: 35±1 kg/m2) who ingested (in randomized order): a) either 0.6 g of whey protein (containing 0.0684 g of leucine) per kg fat‐free mass (28.1±1.0 g whey protein total) or placebo or b) 0.0684 g leucine per kg fat‐free mass (3.3±0.1 g leucine total) or placebo during the 4‐h clamp. Whey protein ingestion significantly reduced the rate of glucose disposal during insulin infusion (2786±324 vs. 3022±383 µmol/min, respectively, P=0.04) whereas leucine ingestion had no effect on insulin‐mediated glucose disposal (2787±212 vs. 2805±238 µmol/min, respectively, P=0.90). We conclude that: a) protein ingestion impairs insulin‐mediated glucose disposal and b) the insulin desensitizing effect of protein ingestion is not mediated by increased leucine availability.

Grant Funding Source: Supported by grants from the NIH and an Ajinomoto Exploratory Research Grant.
Adipocyte specific VEGF deficient mice show diminished endurance exercise capacity (1162.11)
Authors: Zachwieja Nicole , Stricker Janelle , Allen Jessica , Vona‐Davis Linda , Olfert I. Mark
Published: June 2021

Reducing vascular endothelial growth factor (VEGF) in adipose tissue alters adipose vascularity and metabolic homeostasis. We hypothesized that this would also affect metabolic responses during exercise‐induced stress, and that adipocyte‐specific VEGF deficient (adipoVEGF‐/‐) mice would have impaired endurance capacity. Endurance exercise capacity in adipoVEGF‐/‐ (n=11) and littermate control (n=11) mice was evaluated every 4 weeks between 6 & 24 weeks of age using a submaximal endurance run to exhaustion at 20 m/min, 10‐degree incline. Maximal running speed, using incremental increases in speed at 30 second intervals, was tested at 25 weeks of age. Beginning at 6 weeks, and continuing with all time points, endurance run time to exhaustion was 30% lower in adipoVEGF‐/‐ compared to controls (p<0.0001). The age‐associated rate of decline in endurance capacity was similar in adipoVEGF‐/‐ vs. control mice (36.7 ± 8.6% vs. 36.6 ± 10.6%), and there was no difference in maximal running speed between the groups (36.0 ± 1.6 vs. 34.2 ± 0.9 m/min). A decrease in endurance capacity without a change in maximal exercise capacity suggests that lack of adipose VEGF and/or reduced adipose capillarity can limit the utilization of adipose energy stores during exercise. Future studies will determine if VEGF directly influences fatty acid mobilization or if these effects are due solely to changes in adipose capillarity.
AMPKα2 skeletal muscle activity is required to induce the physiological effects of insulin on fatty acid uptake (1162.3)
Authors: Abbott Marcia , Koulakis Anastasia , Kern Marialice , Turcotte Lorraine
Published: June 2021

Exercise training has been deemed a beneficial treatment for obesity and insulin resistance induced by a high fat diet. Further, exercise training may modulate its metabolic effects via AMPK activation, a key signaling molecule in metabolic regulation. It is widely accepted that insulin stimulation (IS) results in an increase in fatty acid uptake (FAU) in skeletal muscle. The purpose of this study was to determine whether AMPKα2 activity in skeletal muscle is required for the benefits of exercise training in regards to insulin sensitivity while under high fat‐fed conditions. Wild type (WT) and AMPKα2 dominant negative (DN) male C57BL/6 mice were divided into control diet ± voluntary wheel running (CD, n=10; VWR, n=10) or high fat diet (60% fat Bio‐Serv) ± VWR (HFD, n=10; VWR+HFD, n=10). After 6 wks, hindlimbs were perfused (550μM palmitate, 6mM glucose, [1‐14C]palmitate, and 450μU/ml insulin). As expected, IS increased FAU (45%) in CD WT mice (P<0.05). However, IS decreased FAU by 35% (P<0.05) in CD DN mice. With VWR, insulin maintained its ability to increase FAU (26%) in WT mice (P<0.05). In DN mice, VWR did not improve the actions of insulin on FAU (39% reduction; P<0.05). When VWR was performed under HFD conditions, the added metabolic stress reduced the ability of insulin to increase FAU (P>0.05) in WT mice. In DN mice, VWR combined with HFD did not change the actions of insulin on FAU (32% reduction; P<0.05). AMPKα2 signaling appears to be required to induce the physiological effects of insulin on FA metabolism. VWR did not improve the aberrant FAU responses to IS in mice lacking skeletal muscle AMPKα2. Further, it appears that VWR is not sufficient to rescue or alter the effects of HFD on insulin‐mediated FA metabolism. Overall, AMPKα2 activity is a necessary component in the regulation of skeletal muscle FA metabolism.
Resveratrol and SRT1720 lead to differential effects in key metabolic organs (1162.5)
Authors: Svensson Kristoffer , Schnyder Svenia , Handschin Christoph
Published: June 2021

Resveratrol (RSV) and SRT1720 (SRT) are postulated calorie restriction mimetic compounds, and thus are suggested to be potential pharmaceutical interventions against obesity and related metabolic complications in humans. The metabolic effects of these two compounds have been proposed to be mediated by the transcriptional co‐activator, peroxisome proliferator‐activated receptor γ co‐activator 1α (PGC‐1α), although the exact requirements for PGC‐1α in mediating these effects is still not known. Here we investigate the role of skeletal muscle PGC‐1α in mediating whole body and muscle‐specific adaptations to RSV and SRT treatment during high fat diet feeding in mice. Furthermore, we also perform a comparative analysis of the effects of these two individual compounds on metabolic processes in key metabolic organs. Using mice with a skeletal muscle‐specific deletion of PGC‐1α, we can demonstrate that PGC‐1α is necessary for transcriptional induction of mitochondrial genes in skeletal muscle, while it is dispensable for the systemic metabolic effects of both RSV‐ and SRT‐treatment. We can furthermore demonstrate both analogous and differential effects of RSV‐ and SRT‐treatment on lipid metabolism and mitochondrial biogenesis in liver as well as adipose tissue. These data provide novel insight into the metabolic effects of RSV and SRT, and thus highlight both beneficial and potential detrimental effects of these compounds when used as a therapeutic intervention for obesity.

Grant Funding Source: Supported by: SNSF ‐ Swiss National Science Foundation
High protein diet and insulin effects on catabolism in rats with burns and disuse (1162.8)
Authors: Baer Lisa , Wu Xioawu , Wolf Steven , Wade Charles
Published: June 2021

Severe burn and disuse result in metabolic alterations associated with systemic inflammatory and endocrine changes. Such changes include extreme inflammation, hypermetabolism and catabolism, resulting in dramatic endocrine shifts to enable the body to adapt to the severe burns. Using our rodent model of burn and disuse, daily insulin demonstrated improvements in total muscle and fat mass and reduced catabolism. To determine if diet can further improve these outcomes, we examined the effects of a high protein diet plus daily insulin on lean mass, blood urea nitrogen (BUN) and nitrogen balance. Male rats assigned to four groups: 20% or 40% protein diet, with or without insulin (n=6/group). Diet caloric content remained constant. Rats received a 40% TBSA scald burn and then hindlimb unloaded. Food intake and urine were collected daily for 14 days after injury. BUN levels were significantly reduced in the 20% group. A decreased nitrogen balance was observed in the 20% groups at day 3 with a steady increase over time, but remained in negative balance. The 40% groups showed a reduction by day 3 and then were in positive balance by day 14. Increased dietary protein did not improve lean, fat or muscle mass. Increased dietary protein coupled with daily insulin failed to mitigate muscle loss or catabolism in response to burn injury and disuse.

Grant Funding Source: Supported by: US Army MRMC & Juvenile Diabetes Foundation
Paracrine effects of cancer on skeletal muscle: myosin loss and mitochondrial dysfunction (1163.10)
Authors: McLean Julie , Moylan Jennifer , Andrade Francisco
Published: June 2021

Cancer cachexia is a severe skeletal muscle wasting syndrome directly responsible for 30% of all cancer deaths. Cancer exerts paracrine effects on skeletal muscle that trigger myosin loss and metabolic dysfunction. Clinical trials using cyclooxygenase 2 (COX2) inhibitors to treat cachexia had some success. Therefore, we hypothesized that tumor‐induced COX2 expression contributes to myosin loss and mitochondrial dysfunction. We incubated C2C12 myotubes with control or Lewis lung carcinoma conditioned medium (LLCCM) for 48hr. We measured protein content by western blot, and oxygen consumption rate (OCR) of intact myotubes using a Seahorse XF24. Protein content is presented as means±SD in arbitrary units (n=6/group, t test). OCR is presented in pmoles O2/min (n=8/group, repeated measures ANOVA). LLCCM treatment decreased myosin content by 70%, from 7.2±2.7 to 2.1±1 AU (p<0.01), while COX2 content increased 75%, from 1.9 ±0.1 to 3.3 ±0.2 (p<0.001). There was no significant effect on mitochondrial complex content, p >0.05 for complexes I, III, IV and V. LLCCM decreased basal OCR by 30% (p<0.003), but did not alter leak or maximal uncoupled OCR. We found that LLCCM treatment induced myosin loss and impaired mitochondrial function in C2C12 myotubes, correlating with increased COX2 content. These data support our original hypothesis, and suggest that interventions that target COX2 may be useful for cancer cachexia.

Grant Funding Source: Supported by Support: NIH AR055974 (FHA).
Simultaneous reversal of age‐related declines in muscle health and cognition with transplantation of preconditioned mesenchymal stem cells (1163.2)
Authors: Huntsman Heather , De Lisio Michael , Kolyvas Emily , Merritt Jennifer , Bhattacharya Tushar , Jensen Tor , Rhodes Justin , Boppart Marni
Published: June 2021

Mechanical strain can potently influence paracrine factor release from skeletal muscle‐derived mesenchymal stem cells (mMSCs). Since the MSC secretome has the potential to impact both local and distant tissue health, we assessed the extent to which transplanted mMSCs influence the local muscle environment as well as brain plasticity and behavior. mMSCs isolated from young muscle were preconditioned (10% multiaxial strain, 5 hrs) and immediately transplanted into the muscle of 24 month old mice. Mice receiving saline or non‐strained mMSCs served as controls. In addition to measuring indices of muscle health, the dentate gyrus of the hippocampus was evaluated for immature neurons one week post‐injection. Two weeks after transplantation, cognitive performance was measured by water maze, rotarod, and contextual fear conditioning. Although satellite cell number and fiber size were unchanged, vessel size and number of neuromuscular junctions were greater in muscle injected with strained mMSCs versus controls resulting in increased muscle function (P < 0.05). A significant increase in the number of immature neurons (P < 0.05) and a trend towards increased cognitive function on the water maze test was observed in these mice. Overall, mMSC preconditioning prior to transplantation may provide a novel method to prevent or reverse age‐related impairments in muscle strength, neuroplasticity, and cognitive function.

Grant Funding Source: Supported by Ellison Medical Foundation (AG‐NS‐0547‐09), Abbott Laboraties (Abbott CNLM ZA68)
Antagonizing PPARγ improves muscle fiber force production and reduces lipid infiltration following chronic muscle injury (1163.20)
Authors: Gumucio Jonathan , Flood Michael , Roche Stuart , Mendias Christopher
Published: June 2021

Following chronic tears, certain muscle groups develop an accumulation of ectopic fat within muscle fibers, as well as in plaques within the extracellular matrix. Muscle atrophy, fibrosis and inflammation often accompany this fat infiltration, and these changes are commonly referred to as “fatty degeneration.” As PPARγ is a potent inducer of adipogenesis, we tested the hypothesis that antagonizing PPARγ signaling would reduce fatty degeneration and increase muscle fiber specific force production in a rat full‐thickness severe rotator cuff tear model. Rats were subjected to a full thickness tenectomy of the supraspinatus and infraspinatus tendons and received either standard chow or chow infused with the PPARγ antagonist GW9662. Thirty days after tear, muscles were harvested and prepared for muscle fiber contractility, histology, and RNA isolation. Compared with control rats, torn muscles from PPARγ antagonist rats exhibited a 15% increase in specific force production. Additionally, antagonizing PPARγ reduced the amount of lipid plaques, along with a downregulation of several mRNA transcripts involved in canonical muscle lipid metabolism, macrophage accumulation, atrophy, autophagy and fibrosis. While further studies are necessary, the results from the current work support PPARγ as a potential therapeutic target for the treatment of fatty degeneration in chronic muscle tears.
Delay in post‐ovariectomy estrogen replacement negates post‐exercise estrogen augmentation of muscle satellite cell population in rats (1163.23)
Authors: Mangan Gary , Hamilton Victoria , Hubbard Andrew , Bombardier Eric , Tiidus Peter
Published: June 2021

17β‐Estradiol (E2) enhances activation, proliferation and differentiation of muscle satellite cells (SCs) following exercise via activation of estrogen receptor‐α (ER) and phosphatidylinositol 3‐kinase (PI3K) signaling. Supplementation of E2 through hormone replacement has been shown to ameliorate muscle atrophic effects of menopause in women. Delay in post‐menopause E2 replacement in humans or following ovariectomy in rodents has resulted in negation of positive health or regenerative effects of E2 on various tissues. To determine if delay in E2 replacement influenced E2 effects on skeletal muscle, 64, nine‐week old, ovariectomized Sprague‐Dawley rats were divided into eight groups based on: E2 status (0.25 mg estrogen pellet or sham), exercise status (90 min run @ 17 m/min, ‐13.5° or unexercised) and E2 replacement (“proximal”; E2 replacement within one week or “delayed”; eleven‐weeks following ovariectomy). Significant increases in total SCs were found in soleus muscle (immunofluorescent co‐localization of nuclei with Pax7) 72 hr following eccentric exercise (p < 0.05). Only “proximal” estrogen‐replacement resulted in further enhancement in total muscle SCs in exercised rats (p < 0.05). Delayed estrogen‐supplementation did not produce a further augmentation in post‐exercise SCs. This suggests the existence of a short window of opportunity where E2 replacement must commence following loss of endogenous estrogen production in order for benefits to skeletal muscle regeneration to manifest.

Grant Funding Source: Supported by NSERC
Muscle protein synthesis, degradation and the effect of mTORC1, after resistance exercise (1163.7)
Authors: West Daniel , Baehr Leslie , Shamim Baubak , Gomes Aldrin , Bodine Sue , Baar Keith
Published: June 2021

We aimed to measure mTORC1 signaling, rates of muscle protein synthesis (MPS), and proteasome activity after acute resistance exercise (REx). A second aim was to determine whether mTORC1 signaling impacted MPS in late recovery. Rats (8 m.o. FBN hybrids) underwent unilateral hindlimb stimulation that eccentrically contracted the tibialis anterior (TA). All measures in the stimulated TA were expressed relative to the contralateral control TA. TA muscles were collected at 1.5, 3, 6, 18, and 36 h after REx, and at 3 and 18 h after REx plus rapamycin treatment to block mTORC1 activity. There was a robust activation of mTORC1, whereby phosphorylation of S6K1 (T389) peaked at 3 h (134±22‐fold inc.) and returned to baseline by 18 h; phosphorylation of S6 (S240/244) peaked at 6 h (26±4‐fold inc.). MPS peaked at 6 h (67±31% inc.) before gradually declining toward baseline through 36 h. Principal proteasome activities, 26S and 20S beta5, tended to decrease (~15‐20% at 6‐12 h), and then increase at later time points (~15‐20% above rest at 18‐36 h). Interestingly, rapamycin treatment, while completely blocking increases in p‐S6K1, only partially blocked the elevation in MPS at 18 h. In summary, REx stimulated a large increase in mTORC1 signaling and a moderate sustained increase in MPS, and inhibiting mTORC1 did not fully abolish the MPS effect at 18 h. Further, for the first time, we have directly measured proteasome activity after REx.

Grant Funding Source: Supported by NSERC and Veteran Affairs (RR&D Merit Grant E7766‐R)
Extracellular ATP as a relevant mediator between skeletal muscle depolarizaition and interleukin‐6 expression: from cell culture to whole animal adaptations (1164.10)
Authors: Buvinic Sonja , Bustamante Mario , Fernández‐Verdejo Rodrigo , Casas Mariana , Vásquez Javiera , Jaimovich Enrique
Published: June 2021

Interleukin‐6 (IL6) is a relevant myokine, highly expressed in skeletal muscle cells upon exercise. We have demonstrated that extracellular ATP is a relevant mediator between skeletal muscle depolarization and intracellular calcium signaling leading to gene expression. The aim of this work was analyze the role of this pathway in controlling IL6 expression, from culture cells to an animal model.

In the L6 skeletal cell line overexpressing Pannexin 1, leading to a rise in ATP release at rest, IL6 expression was 2.5 fold increased. In newborn‐derived rat myotubes, IL6 expression evoked by a tetanic electrical stimulation was abolished when extracellular ATP signaling was blocked by the ectonucleotidase apyrase or by the P2Y/P2X receptors general inhibitor suramin. In myotubes and isolated mice skeletal fibers, both exogenous ATP and ADP increased IL6 expression in a concentration dependent manner. In myotubes, ATP evoked IL6 expression through a pathway involving Ca2+ and IL6 release, activating an autocrine positive loop. In a physiological model of exercise, skeletal fibers from voluntarily active mice (5 weeks voluntarily training in an exercise wheel) had an increased sensitivity to ATP for IL6 expression compared with sedentary mice; ATP sensitivity directly depended on the level of physical activity performed. Considering that increased ATP sensitivity occurs even in the absence of metabolic modifications, ATP sensitivity emerges as an early, highly sensitive marker of muscle adaptation to training. This could have an impact not only in the exercise‐dependent expression of IL‐6 but also in other physiopathological responses.

Grant Funding Source: Supported by Fondecyt‐1110467‐11100454, Conicyt‐79090021, FONDAP‐15010006, ACT‐1111
Influence of sympathetic vasoconstriction on muscle blood flow control during graded handgrip exercise in young and older adults (1165.2)
Authors: Richards Jennifer , Crecelius Anne , Hearon Christoper , Racine Matthew , Larson Dennis , Luckasen Gary , Dinenno Frank
Published: June 2021

With human aging there is a marked increase in resting sympathetic nervous system (SNS) activity, and older adults also exhibit attenuated vasodilation and skeletal muscle blood flow during exercise.

We tested the hypothesis that SNS restraint of muscle (forearm) blood flow (FBF) is greater in older compared with young adults during graded handgrip exercise (5, 15, 25% maximal voluntary contraction (MVC)). FBF was measured (Doppler ultrasound) and vascular conductance (FVC) was calculated in 12 young (21±1 years) and 12 older (62±2 years) adults in control conditions and following local α+β‐adrenergic receptor blockade (intra‐arterial infusions of phentolamine and propranolol). Compared to young, older adults exhibited significantly lower FBF at 15% MVC (216±12 vs. 177±15 ml/min) and 25% MVC (344±24 vs. 277±17 ml/min; both P<0.05). Following local adrenergic blockade, the increase in FBF was similar in young and older adults across exercise intensities, and the age difference in FBF during exercise was similar to control conditions. Our data indicate that, during graded mild‐to‐moderate intensity handgrip exercise, the reduction in muscle blood flow in older healthy adults is not due to augmented sympathetic vasoconstriction, but rather due to impairments in local vasodilatory signaling with age.

Supported by HL095573

Grant Funding Source: Supported by HL095573
Increased brain‐derived neurotrophic factor in lumbar dorsal root ganglia contributes to the exaggerated exercise pressor reflex in heart failure (1165.7)
Authors: Wang Hanjun , Wang Wei , Rozanski George , Zucker Irving
Published: June 2021

An exaggerated exercise pressor reflex (EPR) causes excessive sympatho‐excitation and exercise intolerance during physical activity in the chronic heart failure (CHF) state. Evidence showed that the peripheral sensitization of muscle mechanical afferents (mechanoreflex) is an important contributor to the exaggerated EPR in CHF whereas the metaboreflex is blunted. To date, the mechanisms underlying the selective sensitization of the mechanoreflex in CHF remain unclear. We hypothesized that brain‐derived neurotrophic factor (BDNF) in muscle afferent neurons plays a critical role in the sensitization of the mechanoreflex in CHF. Increased BDNF expression was observed in lumbar DRGs from CHF rats compared to sham rats (8.3±1.1 vs. 19.5±1.8 pg/g, n=6, P<0.05). Immunofluorescence further showed a greater increase in the number of BDNF‐positive neurons (mainly mechanically sensitive, marked by NF200) in CHF rats. Finally, chronic epidural delivery of anti‐BDNF (10 µg/ml) to the lumbar DRGs attenuated the exaggerated pressor response to either static contraction (EPR activation) or passive stretch (mechanoreflex) in CHF rats whereas this intervention had no effect on the pressor response to hindlimb arterial injection of capsaicin (metaboreflex activation). These data suggest that increased BDNF in lumbar DRGs contributes to the exaggerated EPR by sensitizing the mechanoreflex in CHF rats.

Grant Funding Source: American Heart Association (#12SDG12040062)
Acute hypoxia rapidly alters myotube size in vitro and myostatin signalling in vivo (1167.1)
Authors: Elliott Bradley , Renshaw Derek , Getting Stephen , Watt Peter , MacKenzie Richard
Published: June 2021

Skeletal muscle loss correlates with mortality across chronic pathological disorders. Chronic O2 reduction induces atrophy in healthy mountaineers and hypoxemic COPD patients. Hypoxic rats and hypoxemic COPD patients show elevated myostatin expression, a regulator of muscle protein turnover and satellite cell proliferation. We hypothesized hypoxia induces atrophy via myostatin signalling.

C2C12 myoblasts (N = 3) were scratched and exposed to 18 hrs of 21% or 1% O2 +/‐ 10 µM PS145 (NF‐κB inhibitor) to measure migration. Myotubes (N = 3) were exposed to 2, 24 or 48 hr of 21% or 1% O2 +/‐ 10 µM PS145 to measure atrophy. Healthy males (N = 9) were exposed to 12% O2 for 2 hours with muscle tissue and plasma collected pre‐ and post‐exposure. Protein expression was quantified by Western blot (muscle) or ELISA (plasma).

Hypoxic myoblasts showed reduced migration. Hypoxic myotubes showed atrophy at 2 hr, which increased at 24 and 48 hr. Hypoxic impairment of myoblast migration and myotube atrophy was offset by PS1145 incubation. Hypoxic participants showed reduced muscle myostatin, and trends for increased ubiquitin binding and NF‐κB activity. Plasma myostatin was unchanged.

Here we demonstrate hypoxia rapidly induces atrophy in vitro via NF‐κB dependent mechanisms. Hypoxia alone induces atrophy, independent of other factors, and an atrophic environment in vivo similar to that seen in chronic hypoxic disorders, such as COPD.

Grant Funding Source: Supported by The Society for Endocrinology (UK)
Muscle adaptation in obese rats exposed to chronic hypoxia (1167.3)
Authors: Gamboa Jorge , Macarlupu Jose , Anza Cecilia , Rojas Percy , Villafuerte Francisco
Published: June 2021

Chronic hypoxia decreases fasting glucose and increases glucose utilization in lean and obese rats. Hypoxia increases insulin sensitivity in obese rats, but insulin resistance remains higher compared to lean rats exposed to hypoxia (figure). Skeletal muscle plays an important role in glucose metabolism; thus changes in insulin pathway and mitochondrial metabolism may impact skeletal muscle glucose uptake. We evaluated the adaptation of skeletal muscle to chronic hypoxia in lean and obese rats. We hypothesized that obese rats have less mitochondrial protein content and decreased activation of the ERK/MAPK pathway, one of the components of the insulin‐signaling cascade. For this purpose, Sprague‐Dawley rats were fed with normal chow diet or high‐fat diet (45% Kcal from fat) for 24 weeks in a normoxic environment. Rats were then exposed to hypobaric hypoxia (simulated altitude of 4500m) for 3 weeks. Triceps surae muscles were removed at the end of the hypoxia exposure and processed for western blot analysis. Mitochondrial protein content was evaluated using a monoclonal antibody against the subunit α of complex V. Contrary to our hypothesis, obese rats have a higher mitochondrial protein content than lean rats (35% higher, p=0.025). There was no difference in the activation of the ERK/MAPK pathway between the groups. Further studies are required to elucidate how chronic hypoxia affects glucose metabolism in obese rats.

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Grant Funding Source: Supported by FINCyT (No 046‐FINCyT‐PITEI‐2010) and NIH (K23DK100533)
Hypercapnia attenuates parasympathetic influence on heart rate (1169.10)
Authors: Bloom Jacob , Ramirez Jan‐Marino , Garcia Alfredo
Published: June 2021

Hypercapnia is readily recognized as an important stimulus for breathing and is often encountered during cardio‐respiratory conditions such as sleep apnea and SIDS. This on‐going study uses an in vivo approach to examine how hypercapnia influences parasympathetic tone over heart rate and cardiorespiratory coupling. Optogenetic experiments were conducted in mice expressing channelrhodopsin 2 under the regulation of the ChAT promoter. The nucleus ambiguus (NA) was photo‐stimulated (λ=470nm) while breathing 100% O2 or 95% O2/5% CO2 (hypercapnia). During both 100% O2 and hypercapnia, the respiratory rhythm was not affected during photo‐stimulation of the NA. However, in 100% O2, photo‐stimulation of the NA increased HRV. In contrast, heart rate variability (HRV) was unaffected by photo舉activation of the NA during hypercapnia. Hypercapnia also prevented light‐mediated depression in heart rate. Blockade of β‐receptors, did not affect the ability for hypercapnia to prevent light activated inhibition of heart rate. Thus, hypercapnia appears to attenuate parasympathetic tone over heart rate through a mechanism independent of sympathetic input. These findings have potential implications in understanding the basis of cardio‐respiratory uncoupling found in many conditions such as SIDS and sleep apnea.