bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppression of calcineurin signaling reduces PGC‐1α expression in a chronic model of muscle atrophy
Authors: Roberts Tiffany K , Donaldson Sue K , Gooch Jennifer L , Price S Russ
Published: June 2021

Diabetes mellitus (DM) causes persistent skeletal muscle atrophy, which contributes to renal failure and a decreased quality of life. Contractile activity is known to blunt persistent muscle atrophy, suggesting that decreased calcium‐dependent signaling could contribute to the atrophy process. Therefore, we examined the calcineurin (Cn)/NFAT/MEF2 signaling pathway in a streptocotozin (STZ) –induced DM model of chronic muscle atrophy. Cn protein as well as NFAT and MEF2 transcription activity was decreased in 21d STZ‐DM rats. Additionally, PGC‐1α, a downstream target of Cn/NFAT/MEF2 signaling, was decreased. To determine whether reduced Cn signaling is directly responsible for the decrease in PGC‐1α, we utilized CnA‐isoform specific knockout mice. NFAT and MEF2 activity and PGC‐1α mRNA were decreased in muscle from CnAα−/− and CnAβ−/− mice. PGC‐1α is a transcriptional cofactor with roles in muscle MHCI fiber‐type specification and preservation of muscle mass. In 21d STZ‐DM rats, fiber‐type switching from MHCI to MHCII was observed and MHCII fibers atrophied to a greater extent. These results suggest that atrophy in systemic disease states may be sustained through suppression of Cn signaling and loss of PGC‐1α expression resulting in changes in functional capacity and fiber‐type switching.
Muscle‐Specific Disruption of Sirt1 Reduces Mitochondrial Function and Increases Reactive Oxygen Species Production
Authors: Menzies Keir J , Hood David A
Published: June 2021

Both caloric restriction and resveratrol, a natural polyphenolic compound, have been shown to activate Sirt1, which in turn extends lifespan and increases mitochondrial content and function. Interestingly, the effects of these treatments and those of exercise, rely on many of the same physiological changes. To further examine the relationship between exercise performance and Sirt1 activity, we produced a skeletal‐muscle specific Sirt1‐deficient mouse. These mice were generated using a myosin light chain‐directed Cre‐loxP targeted mutation of the Sirt1 gene. These mice showed no behavioural or phenotypic abnormalities and had similar body and muscle weights as wild‐type (WT) animals. Isolated muscle mitochondria from Sirt1‐deficient mice exhibited a 45% decline in state 4 respiration along with 4‐fold higher production of reactive oxygen species (ROS) in comparison to WT animals. State 3 respiration was also 70% lower, and was accompanied by a 7‐fold higher ROS production. Despite these findings, over a three week period, Sirt1‐deficient mice ran the same average (12 km/day) and total distances on voluntary running wheels. These data indicate that the absence of Sirt1 results in an impaired capacity for mitochondrial ATP synthesis, along with increased ROS production, which may act as a compensatory signal to maintain mitochondrial biogenesis and voluntary exercise performance. Funded by CIHR..
Effects of hibernation‐induced bradycardia on cardiac myosin heavy‐chain and myocardial mRNA expression in grizzly bears
Authors: Barrows Nathan David , Nelson Lynne , Robbins Charles , Rourke Bryan
Published: June 2021

Grizzly bears, Ursus arctos horribilis, tolerate extended periods of low heart rate during hibernation without developing congestive heart failure or chamber dilation. We evaluated myocardial response to hibernation‐induced bradycardia by measuring myosin heavy‐chain (MyHC) isoform expression in the left atria and left ventricles of active and hibernating bears, as well as relative mRNA expression of a set of genes important to muscle plasticity. We supplemented these data with echocardiographic measurements of systolic and diastolic function in unanesthetized bears. Relative MyHC‐α protein expression increased by 10% in the atria during hibernation, uncharacteristic of bradycardia in non‐hibernators. The left ventricle expressed 100% MyHC‐β protein in both groups. Insulin‐like growth factor (IGF‐I) mRNA expression was reduced by ~50% in both chambers during hibernation, consistent with observed ventricular atrophy. A 50% decrease in atrial creatine kinase mRNA expression occurred in hibernators. Taken in context with hemodynamic assessment of increased atrial afterload and ventricular stiffness, these data suggest a potential downregulation of atrial chamber function during hibernation to prevent fatigue and dilation due to excessive work against an optimally filled ventricle.

Supported by NIH MBRS SCORE 2 S06 GM063119, the Autzen Foundation and the WSU Bear Center.
Neural basis of visually evoked head and wing movements in dragonflies
Authors: Grome Natalie Marie
Published: June 2021

Dragonflies make their living by foraging on flying insects. We are using two approaches to study their prey capture behavior. (1) Using close‐up, high speed video, we are recording the head movements of dragonflies (Sympetrum vicinum) to the movement of small moving images that simulate prey items. Our study shows that dragonflies use two distinct types of head/eye movements, tracking and saccades. The former are smooth movements that stabilize the moving prey image. The latter are jerky movements that may be involved in distance estimation. (2) Using intracellular penetration and recording, we are studying a group of neurons that are thought to guide prey capture. By controlling their activity with current pulses we are studying the wing steering movements that they elicit. Injection of a fluorescent dye, Lucifer Yellow, into these neurons allows us to study their dendritic structure within the dragonfly's brain.
Squid branchial hearts: boosting blood pressure in the periphery
Authors: Uyeno Theodore A. , Barbano Duane L.
Published: June 2021

Squids rely on an ancestral molluscan circulatory geometry; a single‐chambered systemic heart and a simple unidirectional flow in which gill and muscle capillaries are in series. However, a fast predatory lifestyle required evolution of traits to meet higher metabolic demands and ensure adequate oxygenation; a closed, high‐ pressure circulatory system and peripheral branchial hearts to boost gill blood pressure. Previous studies have described branchial heart evolution from veins, their use in producing ultrafiltrate, the neural control of contractions, and pulse pressure measurements. However, few studies have investigated branchial heart morphology with a focus on developing functional hypotheses that include considerations of flow and material properties. In this study, we use histology and scanning electron microscopy of corrosion casts to visualize the three‐dimensional orientations of the contractile, tensile, and elastic elements in branchial hearts of Dosidicus gigas (Humboldt squids). The thin‐walled branchial hearts have complex orientations of trabeculae that extend into the lumen. It is thought that the muscle and connective tissue trabeculae are organized to maximize contractile force, to store elastic energy between contractions, to increase luminal surface area, and to decrease heart wall thickness. Study funded by an NAU GBI/TRIF Grant 2008–09.
Effects of IL‐6 receptor inhibition and/or heat treatment on the regeneration of injured mouse skeletal muscle
Authors: Fujita Ryo , Ohira Takashi , Kami Katsuya , Oke Yoshihiko , Nomura Sachiko , Kawano Fuminori , Nakai Naoya , Ohira Yoshinobu
Published: June 2021

Effects of inhibited interleukin‐6 (IL‐6) signal using the rat anti‐mouse IL6 receptor (MR16‐1) with or without heat treatment on the regeneration of injured skeletal muscle was studied in mice. 10‐week‐old C57BL/6JJc1 mice were divided into 5 groups: (1) normal controls, (2) cardiotoxin injection (CTX), (3) administration of MR16‐1 after CTX injection (CTX‐MR), (4) heat treatment after CTX injection (CTX‐H), and (5) administration of MR16‐1 plus heat treatment after CTX injection (CTX‐MR‐H). The CTX (10 μl) was injected into the right tibialis anterior (TA) muscle. MR16‐1 (2 mg) was injected i.p immediately after CTX injection. For heat treatment, the conscious mice were placed in a heat chamber (42 °C) for 60 min 1, 3, and 5 days after CTX injection. Muscles were sampled 7 days after these treatments and cross‐sectional analyses were performed. Regenerating myofibers with centrally located nuclei were noted 7 days after CTX injection. Immunohistochemical analyses revealed that desmin‐ and dystrophin‐positive (+) cells were increased in CTX‐MR and CTX‐MR‐H groups. Further, a number of myogenin + cells were seen in the damaged myofibers of CTX‐H group. These results suggest that blockage of IL‐6 signal and heat treatment facilitate the regeneration of skeletal muscle. This study was supported by Grant‐in‐Aid for Scientific Research S (19100009) from the Japan Society for the Promotion of Science.
Plantaris muscle capillarity is reduced in pulmonary TNFα over‐expressing mice
Authors: Olfert I. Mark , Audet Gerald , Malek Moh , Tang Kechun , Breen Ellen C. , Wagner Peter D.
Published: June 2021

Chronic lung inflammation may contribute to abnormal skeletal muscle microvessel structure and function. To test the hypothesis that elevated pulmonary, and, as a result, systemic tumor necrosis factor‐α (TNFα) levels could contribute to skeletal muscle capillary rarefaction, transgenic mice that over‐express TNFα under the control of the lung‐specific SP‐C promoter were studied. Hind limb muscle mass and capillarity, body weight and exercise capacity in 4–5 month old SP‐C/TNFα (Tg, n=7) and littermate, control (C, n=7) mice were evaluated. Serum TNFα in Tg mice was increased (Tg 29.6±3.6 vs. C 5.2±2.9 pg/ml, p<0.001). Maximal running speed was reduced by 18% in Tg vs. C mice (31±1 vs 38±1 m/min, respectively, p<0.01). Body weight, and heart, gastrocnemius, plantaris and soleus mass were not different between Tg and C groups. Plantaris muscle capillary‐to‐fiber (C:F) ratio was 10% lower in Tg vs. C mice (1.32±0.06 vs. 1.46±0.01, respectively, p<0.05). C:F was not altered in the gastrocnemius (Tg 1.51±0.04 vs. C 1.50±0.02) or soleus (Tg 1.63±0.06 vs. C 1.73±0.01, p=0.15). These data suggest that chronically elevated circulating TNFα may contribute to skeletal muscle pathology in part by capillary rarefaction in some, but not all, hind limb locomotor muscles.
Analysis of dystrophic muscle by two dimensional differential in‐gel electrophoresis
Authors: Dixon Jenna , Gardan‐Salmon Delphine , Lonergan Steven , Selsby Joshua T
Published: June 2021

Duchenne muscular dystrophy (DMD), the most common, fatal, X‐linked disease, is caused by a defect in the dystrophin gene. While dystrophin deficiency is the causative factor in this disease, changes in the expression of other proteins may also contribute to disease pathology. The objective of this investigation was to determine the extent to which dystrophin deficiency impacts the muscle proteome. We performed two dimensional differential in‐gel electrophoresis (2D‐DIGE) (pH 4‐7) on gastrocnemii taken from 6 week old male mice (n=6 mdx; n=6 C57). Of a total of 2200 spots detected, 41 spots (representing unique isoforms or modified proteins) were found to be differentially expressed (p<0.1; 24 spots with p<0.05). Of these, 30 spots were increased and 11 were decreased in mdx compared to control. Using mass spectroscopy and MALDI‐TOF, the identity of the proteins represented was confirmed in 36 spots. These proteins represent a variety of cell functions including apoptosis, mitochondrial function, and cell growth and differentiation. Further analysis of these proteins may allow for the identification of novel pathways and lead to the development of innovative strategies to minimize disease progression.
Intergenic Bidirectional Promoter of Cardiac Myosin Heavy Chain (MHC) Genes in Rats
Authors: Giger Julia M , Baldwin Kenneth M , Haddad Fadia
Published: June 2021

The rat heart expresses two MHC isoforms: beta (b) and alpha (a); these genes are arranged in tandem on the chromosome. We have hypothesized that there is an intergenic bidirectional promoter between these two genes that both promotes transcription of the a isoform and natural antisense of the b isoform. This intergenic bidirectional promoter may hold the key to the coordinated and antithetical regulation of a and b MHC genes that has been long recognized in the field of cardiac muscle regulation. Using a method of in vivo injection of promoter‐reporter plasmids, we have tested promoter activities of intergenic sequence in antisense and sense direction and found that activity requires an intact NF1 site! In fact, NF1 mutation caused a dramatic decrease in activity of the full length alpha promoter, which is very active in rat heart. NF1 site, is not conserved in the sequence of larger mammals. This is especially noteworthy because there is a divergence of b MHC expression in large vs. small mammals: the human and “large” mammals express the b isoform primarily in ventricular muscle whereas the small rodents, eg. the rat, express the a isoform primarily. Yet, both humans and rats express the beta MHC in “slow” fibers of skeletal muscles. To further understand the regulation of isoforms genes, we also tested tissue‐specificity and species specificity of alpha MHC promoter in rat skeletal muscle and heart. Supported by ADA and NIHHBL
Nitric oxide synthase inhibition prevents activity‐induced NFAT nuclear accumulation and skeletal muscle adaptation
Authors: Martins Karen JB , St‐Louis Mathieu , Michel Robin N , Murdoch Gordon K , Dixon Walter T , MacLean Ian M , McDonald Pamela , Putman Charles T
Published: June 2021

Involvement of calcineurin/NFAT signaling in the regulation of skeletal muscle myosin heavy chain (MHC)‐based fast‐to‐slower isoform adaptations (F‐S) is recognized. Less is known about the role nitric oxide (NO) plays in this process.

OBJECTIVE

To investigate the necessity of NO in activity‐induced skeletal muscle F‐S in vivo.

METHODS

Endogenous NO production was blocked by administering L‐NAME (0.75 mg ml−1; ~100 mg kg−1 day−1) during 0, 1, 2, 5 or 10 days of chronic low‐frequency stimulation (CLFS; 10 Hz, 12 h d−1) applied to the tibialis anterior and extensor digitorum longus muscles of rats (L‐Stim; n=6 each group). Control rats received only CLFS (Stim; n=6 each group).

RESULTS

CLFS induced increases in NFATc1 nuclear localization in Stim, which did not occur in L‐Stim at any time point. These results were confirmed by western blot analyses. Moreover, MHC mRNA, protein and fibre type analyses revealed CLFS‐induced F‐S occurred in Stim, but were completely abolished in L‐Stim.

CONCLUSIONS

NO may be regulating activity‐induced MHC‐based F‐S at the transcriptional level via NFAT nuclear accumulation in vivo. *Authors contributed equally. Funded by NSERC, AHFMR, CIHR and CRC.
Specific force generation and injury susceptibility of permeabilized single skeletal muscle fibers from myostatin‐deficient mice
Authors: Kayupov Erdan , Bradley Joshua , Claflin Dennis R , Mendias Christopher L
Published: June 2021

Myostatin is a negative regulator of skeletal muscle mass. Compared with wild‐type (WT) mice, the extensor digitorum longus (EDL) muscles of myostatin‐deficient (KO) mice have greater mass; produce greater maximum isometric force (Fo); exhibit decreased specific force (sFo), defined as Fo normalized by muscle cross‐sectional area (CSA); and sustain greater losses in force following an injury‐inducing lengthening contraction. Our working hypothesis is that the reduced sFo and increased susceptibility to injury observed in whole EDL muscles from KO mice arise from corresponding differences in the constituent fibers. We tested the hypotheses that, compared with WT mice, individual fibers from KO mice have: 1) a reduced sFo and 2) increased susceptibility to contraction‐induced injury. The hypotheses were tested using permeabilized single fibers obtained from EDL muscles. While KO mice had a 21% increase in CSA, the Fo of fibers from WT and KO mice was not different and the sFo of fibers from KO mice was reduced by 18%. There was no difference in force deficits following injury. We conclude that a reduction in the sFo of individual fibers in KO mice contribute to the reduction in sFo at the whole muscle level. Our hypothesis that fibers from KO mice are more susceptible to injury was not supported.
Hypoxic exercise training elicits the mobilization of hematopoietic progenitor cells and endothelial precursor cells into the peripheral blood compartment in healthy men
Authors: Lee Mei‐Yi , Wang Jong‐Shyan
Published: June 2021

Bone marrow‐derived, circulating endothelial progenitor cells (EPC) is contributing to the maintenance of endothelial function and organ perfusion by mechanisms ranging from endothelial repair to postnatal angiogenesis and vasculogenesis. This study investigates how hypoxic exercise training influences the mobilization of EPC subsets into the peripheral blood compartment. Fifteen sedentary healthy men cycled on an ergometer at about 60% of maximal oxygen consumption under 15%O2 condition for 40 minutes per day, 5 days per week for 5 weeks. Our results indicated that acute 12%O2 exercise increased the mobilization of hematopoietic stem cells (CD34+/KDR+/CD117+), early EPC (CD34+/KDR+/CD133+), and endothelial precursor cells (CEP, CD34+/KDR+/CD133−) into the peripheral blood compartment at pre‐training stage. Moreover, both resting and 12%O2 exercise‐mobilized circulating HSC and CEP levels were significantly elevated following 5 weeks of 15%O2 exercise training. However, no significant changes in circulating (CD34+/KDR+/CD31+) and shedding endothelial cells (KDR+/CD31+/phosphotidylserine+) levels occurred following acute or chronic hypoxic exercise. Hence, we conclude that long‐term moderate‐intensity exercise at 15%O2 environment elicits the mobilization of hematopoietic progenitor cells, especially HSC and CEP, into the peripheral blood compartment, which may facilitate angiogenesis or vasculogenesis in exercising muscles.
Peripheral microvascular responses to norepinephrine in women with orthostatic intolerance
Authors: Wenner Megan M. , Taylor Hugh S. , Stachenfeld Nina S.
Published: June 2021

We determined the effects of estradiol (E2) and progesterone (P4) on the cutaneous microvasculature during adrenergic (norepinephrine, NE) stimulation in eight orthostatic tolerant (OT) and eight intolerant (OI) women. We hypothesized that 1) OI have a blunted vasoconstrictor response to NE compared to OT; and 2) that female sex hormones modulate vasoconstriction. A cumulative stress index (CSI) during maximal lower body negative pressure determined orthostatic tolerance. Endogenous E2 and P4 was suppressed with daily administration of a GnRH antagonist (GnRH) for 16 days; we gave E2 (0.2 mg/day, patch) on days 4–16, and P4 (200 mg/day, pills) on days 13–16. We measured skin blood flow (laser Doppler flowmetry) during graded NE infusions (skin microdialysis) during GnRH, E2, and E2+P4. CSI was 871±86 and 397±65 for OT and OI respectively (P < 0.05). The vasoconstrictor response to NE during GnRH was similar between groups (log EC50: OT −3.9, CI −4.9 to −3.1; OI −4.4, CI −5.4 to −3.4). Combined E2+P4 enhanced the vasoconstrictor response in OT (EC50 −4.8, CI −5.6 to −4.1) but was reversed with Ketorolac (−3.8, −4.6 to −3.0). Hormones did not alter vasoconstriction in OI (EC50 −4.3, CI −5.0 to −3.7; −4.9, CI −5.5 to −4.4). Adrenergic response in skin microvasculature was similar between OT and OI. Although OI were insensitive to hormone treatments, P4 augments cutaneous adrenergic responses in OT. HL071159.
Investigation of Sportswear Properties in Athletes Performance in Hot Condition
Authors: YAO Lei , LI Yi
Published: June 2021

Competition in sports is in cut‐into‐edge. How a sportswear enhances athlete's performance in exercise and competition has been illuminated. Properties of sportswear, such as thermal properties, mechanical properties, and liquid water transport properties could play an important role in heat release, comfort and muscle fatigue. To study how a sportswear enhances athletes' performance in hot condition, a systematic study has been designed and conducted to study the influence of sportswear properties on the endurance, core temperature, and metabolism during high intensity exercise. Results show that with specific garment design and fabric selection, sportswear could significantly increase the endurance of performance, delay core temperature increase. The interaction between sportswear and physiology of athletes has been explored. This research is supported by Hong Kong Research Institute of Textiles and Apparel and Hong Kong Polytechnic University through project ‘ITP/014/08TP’.
Effects of ganoderma lucidum spores on expression of PSA‐NCAM and hippocampus mossy fiber sprouting in pentylenetetrazol‐induced epileptic rats
Authors: Li Xiao‐Jie , Wang Shu‐Qiu , Zhang Shi‐Ling , Kang Yu‐Ming
Published: June 2021

The present study was undertaken to investigate the effects of ganoderma lucidum spores (GLS) on expression of polysialic acid neural cell adhesion molecule (PSA‐NCAM) and hippocampus mossy fiber sprouting (MFS) in pentylenetetrazol (PTZ)‐induced epileptic rat model. Thirty male Sprague‐Dawley rats (180–220g) were divided into three groups: control, GLS and epilepsy model groups. The results showed that, compared with epilepsy model groups, the rats of GLS group have longer latent periods, and lower seizure level. Their seizure durations also were shorter than epilepsy model group. In the granular cell layer of dentate gyrus and stratum pyramidal and stratum oriens of the CA3 region, epilepsy model group have more PSA‐NCAM than GLS group and control group; compared with epilepsy model group, MFS in the granular cell layer of dentate gyrus were obviously less in GLS group, and no statistically significant difference was detected when compared with control group; in the stratum pyramidal and stratum oriens of the CA3 region, MFS in GLS group was less than epilepsy model group, but obviously more than control group.

Conclusion

For PTZ induced epileptic rats, there were different severities of MFS in different regions of hippocampus. GLS can decrease expression of PSA‐NCAM and MFS effectively, and that might be one of its mechanism of lighten rats′ seizure induced by PTZ.
Hormetic effects of serum deprivation on androgen regulation of dopamine cell viability
Authors: Cunningham Rebecca L , Singh Meharvan
Published: June 2021

Hormesis is the beneficial response of treatments harmful at high concentrations but at low levels confer resistance, such as ischemic preconditioning and dietary restriction. Androgens have been shown to increase the vulnerability of dopamine neurons via an oxidative stress mechanism. In this study, we examined if serum deprivation alters androgens’ effects on oxidative stress in a dopaminergic cell line (N27 cells). N27 cells were exposed to tert‐butyl‐hydrogen peroxide (500uM) 24hrs prior to androgen exposure in either a serum‐free environment or charcoal‐stripped serum environment. Our results showed decreased neuronal vulnerability to oxidative stress in response to androgens in the serum‐free environment compared to the charcoal‐stripped environment. Indicating that serum deprivation had a hormetic effect on androgen regulation in dopamine neuronal viability. To determine underlying mechanisms, N27 cells in a serum‐free environment were exposed to androgens for varying time periods (0–48hrs) and imaged to determine morphology. Analysis showed increased neurite outgrowth after 2hrs of androgen exposure, indicating increased synaptic plasticity. These results suggest that serum deprivation can confer a hormetic effect on dopamine neuronal viability in response to androgens by increasing synaptic plasticity. Supported by NIH grants F32NS061417‐01 to RLC and AG022550 and AG027956 to MS.
Cannabinoid action on cholecystokinin‐responsive nodose ganglion cells
Authors: Edwards Gaylen L , Page Annie , Harris Meghan D , Hammond Sherri , Freeman Kimberly G
Published: June 2021

It has been demonstrated that fourth cerebroventricular application of cannabinoid agonist facilitates food intake at lower doses than effective doses injected in the lateral cerebroventricle (Physiol Behav 80: 611, 2004). It has also been reported that direct injection of cannabinoid agonist into the lateral parabrachial nucleus facilitates food intake (Am J Physiol 1240: 111, 2008). These data indicate hindbrain endocannabinoid systems are important in the control of food intake. More recently, we have found that cannabinoid agonist injected into the fourth ventricle attenuates the elevation of cFos‐immunoreactivity in the nucleus of the solitary tract after IP cholecystokinin injection (Reddy et al, unpublished). As an extension of those studies, we examined the ability of cannabinoid to suppress cFos activation in nodose ganglion cells to cholecystokinin (CCK). CCK increased cFos expression in nodose ganglion cells. Application of the cannabinoid agonist, CP 55,940 (1μM), suppressed this activation. These data suggest that cannabinoids may be acting on vagal afferent fibers to suppress visceral afferent activity important to ingestive behavior. (Supported by University of Georgia Office of the Vice President for Research)
Glucagon Activates the AMP‐Activated Protein Kinase/Acetyl‐CoA Carboxylase Pathway in Adipocytes
Authors: Peng I‐Chen , Chen Zhen , Hsu Pang‐Hung , Su Mei‐I , Tsai Ming‐Daw , Shyy John Y‐J.
Published: June 2021

Glucagon is important in regulating lipid metabolism, in part through its inhibition of fatty acid (FA) synthesis in adipocytes. Given that acetyl‐CoA carboxylase (ACC) is the rate‐limiting enzyme for FA synthesis, the proposed mechanism is that glucagon activates PKA, which in turn phosphorylates ACC at Ser‐77 and Ser‐1200, thereby attenuates the lipogenic activity of ACC. Because AMPK also inhibits FA synthesis by phosphorylating ACC Ser‐79, we examined the involvement of AMPK in the glucagon‐elicited signaling in adipocytes. Western blot showed that glucagon treatment increased the phosphorylation of AMPK Thr‐172 and ACC Ser‐79 in adipocytes. LC/MS/MS revealed that ACC Ser‐79, but not Ser‐77 or Ser‐1200, was phosphorylated in these cells. The glucagon‐induced ACC Ser‐79 phosphorylation was blocked by pharmacological inhibition or genetic ablation of AMPK. In vitro kinase activity assay showed that Ca2+/calmodulin‐dependent protein kinase kinase β (CaMKKβ) is the upstream kinase phosphorylating AMPK responding to glucagon. As well, CaMKKβ inhibition reduced the AMPK activity with attenuated ACC Ser‐79 phosphorylation. Furthermore, CaMKKβ−/− mice receiving glucagon did not show an activation of the AMPK/ACC pathway in their adipose tissue. Our results suggest that AMPK is crucial in regulating the glucagon‐elicited lipid metabolism in adipocytes.
Activation of Infammasomes by Visfatin in Mouse Endothelial Cells
Authors: Xia Min , Hu Junjun , Wang Zhengchao , Zhang Chun , Li Ningjun , Li Pin‐lan
Published: June 2021

Inflammasomes have been reported to serve as intracellular machinery initiating and promoting the innate immune reaction and inflammatory response to various danger signals. The present study hypothesized that activation of inflammasomes is one of the important mechanisms mediating endothelial inflammatory response to adipokines in obesity. To test this hypothesis, we first characterized the expression of 3 major inflammasome components in mouse endothelial cells (ECs) including cryopyrin (also called NALP3), apoptosis‐associated speck‐like protein (ASC), and caspase‐1. By RT‐PCR and immunocytochemistry, we found that these 3 molecules were abundantly expressed in mouse ECs. Fluorescent confocal microscopy showed that these molecules could be detected in the cytoplasma of mouse ECs, but they were not colocalized under control condition. However, when these cells were incubated with adipokine, visfatin even for two hours, these molecules were aggregated to form a NALP3 inflammasome, indicating the assembling and activation of this inflammatory machinery. Colorimetric measurement of caspase‐1 activity demonstrated that visfatin significantly increased caspase‐1 activity by about 4 folds. These results suggest that activation of inflammasomes may be one of initiating mechanisms responsible for the action of adipokine‐visfatin to induce endothelial dysfunction and inflammatory injury (supported by NIH grant HL057244, HL091464 and HL075316)
Skeletal muscle protein synthesis and mTORC1 signaling following resistance exercise in young and older men and women
Authors: Fry Christopher S , Drummond Micah J , Glynn Erin L , Dickinson Jared M , Gundermann David M , Timmerman Kyle L , Dhanani Shaheen , Volpi Elena , Rasmussen Blake B
Published: June 2021

Resistance exercise (RE) training causes muscle hypertrophy in young men and women, but the anabolic response is usually greater in men. Following RE training in older individuals, men have larger increases in muscle size and strength. We hypothesized the muscle protein anabolic response to an acute bout of RE would be the greatest in young men. Young (28±2yrs) and older (69±2yrs) men and women performed a bout of RE at 70% of their maximal strength. Muscle biopsies were obtained at baseline, 3, 6 and 24h post‐RE. Muscle protein synthesis (MPS) and mTORC1 signaling were assessed with stable isotopic techniques and immunoblotting. Post‐RE MPS increased similarly in young men and women (P>0.05). Preliminary data show MPS rates are lower, with no sex‐based differences, in older individuals following RE (P>0.05). Phosphorylation of S6K1 (Thr389) increased similarly at 3, 6 and 24h post‐RE in the young (P<0.05) and increased similarly at 3 hr post‐RE in older men and women but the response was not as prolonged as in young. Although there are notable age‐related differences, it appears that there are no sex‐based differences in the acute muscle protein anabolic response following a bout of high‐intensity RE. Supported by NIH R01AR049877, P30AG024832 and T32HD07539.
Changes in oxidative modifications of skeletal muscle proteins in old rats fed a high fat diet
Authors: MURAKAMI Hitoshi , GUILLET Christelle , TARDIF Nicolas , SALLES Jerome , BOIRIE Yves , WALRAND Stephane
Published: June 2021

Muscle weight loss during aging results from change in protein turnover, but oxidative modifications of proteins also occur in muscle. In addition, dietary fat promote accumulation of modified proteins in tissues. Therefore, high fat diet in old rats may be associated with change in protein turnover and modifications, contributing to sarcopenia. To elucidate this hypothesis, nitrotyrosine (NT) in quadriceps was measured by Western‐Blot in 6 months and 24 months old rats fed a normal fat (NF) or a high fat diet (HF) for 3 months (adult‐NF (n=7), adult‐HF (n=8), old‐NF (n=7), old‐HF (n=7)). NT was significantly increased in old rats, although there was no effect of dietary fat on NT accumulation. However, in some specific mitochondria proteins (100kDa), NT increase was dependent on aging and dietary fat. Interestingly, NT was strongly negatively correlated with muscle mass (r2=−0.716 (p<0.0001), −0.497 (p<0.01) and −0.699 (p<0.0001)) in sarcoplasma, myofibril and mitochondria, respectively). In addition, NT in specific mitochondria proteins was positively correlated with insulin‐resistance evaluated by OGTT (r2=0.535 (p<0.05)). Therefore, NT in muscle proteins is associated with muscle weight loss during aging but not in HF. In addition, NT in specific mitochondria proteins is associated with insulin resistance induced by a high fat diet.
PRAS40 regulates protein synthesis, cell cycle, proliferation and myogenic differentiation in C2C12 myocytes
Authors: Kazi Abid A , Hong‐Brown Ly , Lang Charles H
Published: June 2021

PRAS40 is an mTOR binding protein which has complex effects on cell metabolism. Our study tested the hypothesis that PRAS40 knockdown (KD) in C2C12 myocytes will increase protein synthesis via up‐regulation of mTOR‐S6K1 pathway. PRAS40 KD was achieved using lentiviruses to deliver shRNA targeting PRAS40 or a scrambled control. C2C12 cells were used as either myoblasts or differentiated to myotubes. KD reduced PRAS40 mRNA and protein content by 80% of time‐matched control values but did not alter the phosphorylation of mTOR substrates, 4E‐BP1 or S6K1, in either myoblasts or myotubes. No change in protein synthesis in myotubes was detected. In contrast, protein synthesis was reduced 25% in myoblasts as measured by the incorporation of 35S‐methionine. PRAS40 KD in myoblasts also decreased proliferation rate with increased percent of cells retained in G1 phase. PRAS40 KD myoblasts were larger in diameter and had a decreased rate of myotube formation as assessed by myosin heavy chain content. Immunoblotting revealed 25–30% decrease in total p21 and phosphorylated Rb protein considered critical for G1 – S phase progression. Reduction in protein synthesis was not due to increased apoptosis as cleaved caspase‐3 and DNA laddering were not different between groups. Our results suggest that a reduction in PRAS40 specifically impairs myoblast protein synthesis, cell cycle, proliferation, and differentiation to myotubes.
Tricellulin in Crohn's disease and ulcerative colitis
Authors: Krug Susanne M , Bojarski Christian , Fromm Anja , Schulzke Jörg Dieter , Fromm Michael
Published: June 2021

The tight junction (TJ) protein tricellulina (TRIC) is predominantly located in tricellular TJs (tTJ), the meeting points of three cells. There it forms a barrier to macromolecules without affecting ion permeability. However, at low TRIC levels the tTJ forms a pathway for macromolecules. We studied the pathologic relevance of TRIC expression in Crohn's disease (CD) and ulcerative colitis (UC).

Methods

Dilution potentials and permeabilities for fluorescein and FITC‐dextran 4K were measured with the Ussing technique in sigmoid colon biopsies from controls, active CD, and UC patients. Epithelial and subepithelial resistances were determined by impedance spectroscopy. Expression of TRIC and other TJ proteins were analyzed by Western blot.

Results

Expression of TRIC was reduced in sigmoid colon of UC patients, but not in CD samples. This was in parallel to known changes in expression of e.g. claudin‐4. UC specimens exhibited decreased epithelial resistance, increased subepithelial resistance, and unchanged transepithelial resistance and Na+ and Cl− permeability. In contrast, permeabilities for fluorescein and FITC‐dextran 4K were increased.

Conclusion

TRIC is downregulated in UC, while it is not influenced in CD. As TRIC limits the macromolecular passage in tTJs, its downregulation may contribute to the increased macromolecular permeability in UC. (Supported by DFG FOR 721)
Expression of the Crohn's disease candidate gene, PTPN2, is upregulated in active Crohn's disease
Authors: Scharl Michael , Weber Achim , Hausmann Martin , Rogler Gerhard , McCole Declan F
Published: June 2021

We have recently shown that IFNγ, a major cytokine involved in Crohn's disease (CD), increases expression and activity of the CD candidate gene, PTPN2, in intestinal epithelial cells (IEC). We have also shown that PTPN2 restricts IFNγ‐induced IEC permeability. Here, we investigated whether PTPN2 expression is altered in active CD. mRNA analysis was performed by real‐time PCR. Immunostaining was performed on formalin‐fixed mucosal biopsies. We investigated PTPN2 mRNA levels in biopsies from macroscopically non‐inflamed areas of the terminal ileum, colon or rectum of CD patients in clinical and macroscopic remission; macroscopically inflamed areas of the terminal ileum and colon of CD patients with clinically active disease; or healthy control subjects undergoing routine screening. We observed significantly elevated PTPN2 mRNA in active CD (p<0.05; n=9), but not in CD patients in remission. Immunostaining showed elevated epithelial and lamina propria PTPN2 in CD vs. controls. CD patients with active disease, but not those in remission, also showed an approximately three‐fold increase in tissue IFNγ mRNA levels compared to healthy controls. H&E‐stained biopsy specimens indicated acute inflammation in patients with active CD only. Overall, our findings demonstrate altered PTPN2 expression in active CD and correlate with observed effects of IFNγ on PTPN2 expression in vitro.

Supported by the CCFA and DFG.
SREBP‐2 Activation is Independent of FPP Levels in Neural Cells
Authors: Igbavboa Urule , Eckert Gunter P. , Strandjord Dana M. , Hoff Gero P. , Mueller Walter E. , Wood W. Gibson
Published: June 2021

We recently reported that FPP and GGPP levels and gene expression of FPP synthase and GGPP synthase were significantly elevated in frontal cortex gray and white matter of Alzheimer disease patients as compared with normal neurological controls whereas cholesterol levels and HMG‐CoA reductase gene expression were unchanged. Stimulation of FPP synthase gene expression can occur with the binding of the transcription factor sterol regulatory element binding protein‐2 (SREBP‐2). Activation of the SREBP pathway is induced by low cholesterol levels and low activity or inhibition of that pathway occurs when cholesterol levels are normal or elevated, respectively. However, what is not known is if SREBP‐2 can be activated by changes in FPP levels when cholesterol levels are unaltered as in the case of AD. To begin to understand FPP and GGPP regulation in brain, effects of inhibition of FPP synthase by a direct inhibitor and inhibition of HMG‐CoA reductase by a statin on translocation of SREBP‐2 into the nucleus were examined in mouse primary neurons and SH‐SY5Y neuroblastoma cells. Cells were treated with simvastatin or alendronate for 48 h and protein levels determined by Western analysis. Simvastatin‐induced inhibition of HMG‐CoA reductase significantly increased SREBP‐2 abundance in the nuclear fraction of cells. In contrast, inhibition of FPP synthase which reduces FPP levels but not cholesterol did not stimulate movement of SREBP‐2 into the nucleus. SREBP‐2 activation may not be driving the increase in FPP levels in AD brain. A consequence of the increase in FPP and GGPP in AD patients could be an over‐abundance of prenylated proteins which could certainly exacerbate cell dysfunction. Supported by grants from the National Institutes of Health AG‐23524, AG‐18357 and the Department of Veterans Affairs.
Purification of ACAT Protein
Authors: Gardner Oneil Nicohlas , Chang Catherine C.Y , Chang Ta‐Yuan
Published: June 2021

Acyl‐coenyzme A:cholesterol acyltransferase (ACAT‐1) is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the formation of cholestryl esters from cholesterol and long‐chain fatty acyl coenzyme A. Recently, we employed human embryonic kidney 293 (HEK293) cells to stably express the recombinant human ACAT‐1 protein bearing a six‐hisitidine tag at the N‐terminus and a Flag‐tag at the C‐terminus. The incorporation of the six–histidine and flag‐tag into the ACAT‐1 sequence allows the ACAT‐1 fusion protein to be purified using two affinity columns. This enzyme was purified by solubilizing the cell membrane with 1M of potassium chloride and 3‐[(3‐cholidopropyl)dimethylammonio]‐1‐propansulfanate (CHAPS). The lysate was then loaded onto an immobilize nickel column followed by a monoclonal Flag antibody column. The flag column allowed us to elute the fusion protein by the flag peptide. More importantly, the elution of the fusion protein using the Flag peptide is one of the most critical steps in the purification because it eliminates the use of low pH (pH 3 to pH4) elution conditions that were used in earlier purifications using ACAT monoclonal antibodies. This new purification scheme allows us to recover a larger quantity of active ACAT‐1 protein for further study of the structure and function of the human ACAT‐1 protein. Supported by NIH T34GM008411
Quantification of serum fucosylated glycoproteins as lung adenocarcinoma biomarkers using AlphaLISA technology
Authors: Chow Lu‐Ping , Wen Chu‐Ling
Published: June 2021

Lung cancer is the leading cause of cancer‐related death in Taiwan for recent years. At the time of diagnosis, most of the lung cancer patients are at advanced stages; therefore, new diagnostic strategies detecting the early stage cancer have been eagerly expected. There is increasing evidence that glycosylation plays a significant role in biological processes including oncogenic transformation and metastasis. Several reports suggested that the alterations in protein glycosylation might be associated with the development of certain types of human cancer.

The specific aim of our study is to setup a platform to screen and identify serum glycoproteins as biomarkers of lung cancer. To enrich N‐linked glycoproteins from serum samples, Con A affinity chromatography were established. The captured glycoproteins were tryptic digested and then analyzed by LC‐MS/MS. For high through‐put screening, we established an AlphaLISA assay, which is a bead‐based proximity assay, to quantify the fucosylation level of these glycoproteins.

Comparison of the serum glycoproteom of normal controls and adenocarcinoma patients revealed some cancer‐related proteins. In addition, the changes of composition and amount of the N‐linked oligosaccharide were evaluated in the sera of patients by lectin staining and AlphaLISA.
Mutations in the insulin pathway affect lipid metabolism, brain function and visual physiology in Drosophila melanogaster
Authors: Murillo‐Maldonado Juan M. , Sánchez‐Chávez Gustavo , Salceda Rocío , Riesgo‐Escovar Juan R.
Published: June 2021

Insulin growth factor signaling is involved in growth and homeostasis in mammals. In Drosophila melanogaster, insulin‐like peptides likewise regulate these processes through the evolutionarily conserved insulin pathway. Here, we studied the effects of mutant conditions for insulin pathway genes. We measured total lipid and carbohydrate levels, brain function as assayed by cholinesterase activity levels (acetylcholine is the main brain neurotransmitter in flies), and retinal function by electrorretinograms (ERGs). We found that mutant flies had generally abnormal high levels of lipids, whereas carbohydrates levels were normal. Several mutants tested (mutations in the insulin receptor, chico (the Drosophila insulin receptor substrate), and protein kinase B) also show electroretinogram defects (reductions in amplitude) and a lower cholinesterase activity in head extracts. In general, metabolic and nervous system alterations correlated in the mutants, suggesting that in flies, as is true in humans, altered metabolism may lead to nervous system impairments. CONACYT 162563; 81864.
Anti‐cancer drug F‐59 regulates glucose metabolism through AMPK
Authors: Song Parkyong , Kim Jong Hyun , Suh Phann‐ghill , Ryu Sungho
Published: June 2021

AMP‐activated protein kinase has been described as a key signaling protein which can regulate whole body energy homeostasis. AMPK enhances catabolic pathway such as glucose and fatty acid metabolism in response to increased energy deprivation and switches off protein, carbohydrate and lipid biosynthesis. To identify novel chemical agonist of AMPK, we carried out drug library screening through cell based system. As a result, anti‐cancer drug F‐59 phosphorylates AMPK and downstream substrate ACC in 3T3L1 adipocytes and L6 myoblast. Glucose uptakes are induced by F‐59 alone treatment and co‐treatment with insulin synergistically in differentiated L6 myotube. The inhibition of AMPK by chemical inhibitor, compound C abolishes F‐59 mediated glucose uptake. Furthermore, administration of F‐59 to mouse significantly decreased the fasting plasma glucose concentration with does dependent manner. And finally, we characterized that AMPK can be modulated by reactive oxygen species (ROS) and free radical scavenger, N‐acetylcystein (NAC), reversed the effects of F‐59 on glucose levels. This could suggest that F‐59 can regulate glucose homeostasis in vivo and represent therapeutic potential in Type 2 diabetic model.
Temporal Gene Expression Profiling of Bone Repair in a Rat Calvarial Defect
Authors: Siller‐Jackson Arlene J , Bowman Phillip D , Wenke Joseph C , Bynum James A , Hammamieh Rasha
Published: June 2021

Critical size defects (CSD) fail to heal and regenerate bone while noncritical defects produce an acceptable repair after bone loss. To improve outcome following significant bone loss we sought to identify genes differentially expressed between a 4 mm (noncritical) and 8 mm (critical) size defect in a rat calvarial model. Calvarial defects were created in 12 week‐old rats (6 per group) and RNA was extracted from the defect at 1, 3, 5, 7, 10, 14 and 21 days after injury. Following labeling and hybridization to Agilent rat whole genome microarrays, analysis with GeneSpring® software indicated 3,564 genes were significantly altered in their expression over all time points, 299 were unidentified genes and ESTs. Gene ontology and functional classification of genes altered in expression between the CSD and non‐CSD identified 122 statistically significant, differentially expressed genes related to bone and muscular development, function, and disorders. Microarray findings were validated with RT‐PCR. By reference to Ingenuity Pathway Analysis many of these 122 genes were related to the canonical Wnt/β‐catenin and Runx2 signaling pathways. Knowledge acquired through analysis of gene activity at each stage of bone healing and non‐healing is contributing to our understanding of bone repair and regeneration, and may provide the basis for healing critical size defects.
Site‐Specific Reciprocal Modification of Human IRS‐2 by O‐GlcNAcylation or Phosphorylation
Authors: Ball Lauren Elizabeth , Berkaw Mary Neale
Published: June 2021

Insulin receptor substrates 1 and 2 (IRS‐1/2) are homologous adaptor proteins that mediate differential metabolic effects of insulin and insulin like growth factor‐1 (IGF‐1). Tyr phosphorylation of IRS proteins by the insulin/IGF‐1 receptors creates interaction motifs for binding partners and subsequent activation of the PI3K/Akt and Grb2/MAPK pathways. IRS protein interactions are further modulated by Ser/Thr phosphorylation in response to nutrient excess and stress. To determine the impact of nutrient sensitive Ser/Thr O‐GlcNAc modification of IRS‐2 on protein interactions and the potential interplay between phosphorylation and O‐GlcNAcylation, the modifications of human IRS‐2 were characterized by tandem mass spectrometry. The protein was immunoprecipitated from HEK293 cells grown in the presence of the O‐GlcNAcase inhibitor, PUGNAc. The gel purified protein was proteolytically digested and the peptides were separated and analyzed by nRP‐LC‐IT‐MS utilizing fragmentation by ETD and CID. This analysis has revealed four novel sites of O‐GlcNAc modification which are distributed throughout the IRS‐2 molecule. Two of the O‐GlcNAcylated residues are known sites of phosphorylation and may represent sites of reciprocal regulation by a kinase and the O‐GlcNAc transferase. Similar to previously characterized human IRS‐1, IRS‐2 is O‐GlcNAc modified within a C‐terminal domain shown to interact directly with the insulin and IGF‐1 receptors. These findings will facilitate determination of the functional effects of O‐GlcNAc modification on IRS‐2 mediated insulin and IGF‐1 signaling.