bluuebunny/crossref_metadata_2025 huggingface dataset:
Three Hours of Intermittent Hypoxia in Young Healthy Humans Induces Electrocardiographic Changes
Authors: Baker Sarah E. , Newhouse Lauren P. , Joyner Michael J. , Curry Timothy B. , Limberg Jacqueline K.
Published: November 2023

Background

Individuals with sleep apnea experience repeated intermittent hypoxic exposures during sleep. Previous work in sleep apnea showed patients have an increased heart rate (HR), corrected QT interval (QTc), QTc dispersion and P‐wave maximum duration and dispersion (max‐min P‐wave duration). Such changes have been linked to increased risk of cardiovascular disease and sudden cardiac death. Unfortunately, the mechanisms contributing to these changes are not completely understood. Given that individuals with sleep apnea experience repeated intermittent hypoxic exposures during sleep, we tested the hypothesis that exposure to intermittent hypoxia (IH) in young healthy adults would result in similar ECG changes to those seen in patients with sleep apnea.

Methods

To test this hypothesis, we completed ECG analysis of 7 healthy humans (6M/1F, Age=29±5 years, BMI=24±4kg/m2) without sleep apnea before and after 3‐hours of exposure to IH. Subjects were exposed to hypoxic gas (0.05 FiO2) for 25‐seconds separated by 2‐minutes of room air breathing. This resulted in 25 hypoxic events per hour in which oxygen saturation was significantly reduced (Normoxia: 97%, Hypoxia: 89%, p<0.01). ECG analysis was completed in Lab Chart 7 software and consisted of measurement of intervals (RR, PR, QT), durations (P, QRS), and amplitudes (P, Q, R, S, T) during 10 minutes of normoxic quiet rest before and after IH exposure. QTc was calculated using Bazett's formula (QT duration/√HR). Dispersion of QTc and P waves were calculated as max duration ‐min duration.

Results

HR was significantly increased following IH (60±8 vs. 66±8 beats/min, baseline vs. post‐IH, respectively, p<0.05). There was no significant change in P‐wave maximum duration or dispersion following IH. QT interval was significantly decreased and QTc was significantly increased following IH (QT interval: 377±13 vs. 368±8ms, QTc: 377±22 vs 384±22ms, baseline vs. post‐IH, p<0.05 for both). QTc dispersion was not significantly different from baseline following IH.

Conclusions

We found that 3 hours of experimentally‐induced IH in young healthy individuals resulted in increased HR, decreased QT interval, and increased QTc. These data suggest that IH exposure experienced by patients with sleep apnea may contribute to ECG changes commonly observed in this population and subsequent increases in cardiovascular morbidity and mortality.

Support or Funding Information

Funding: DK90541 (MJJ), HL120570 (JKL)
mdx Mice are Protected From Diet‐Induced Obesity Possibly Due to Increased Skeletal Muscle Energy Expenditure
Authors: Kwon Frenk , Gamu Daniel , Tupling Allan Russell
Published: November 2023

mdx mice, the murine model of Duchene muscular dystrophy, are characterized by aberrant skeletal‐muscle Ca2+ handling and increased whole body energy expenditure. Increasing skeletal muscle energy expenditure is one mechanism that may protect against excessive weight gain and diet‐induced obesity. mdx mice have previously been shown to resist excessive adiposity when fed a diet moderately high in fat (16% kcal fat). However, it is not clear if this protection persists when they are provided with a typical Westernized high‐fat diet (HFD). We hypothesized that despite consuming a diet higher in fat (42% kcal fat), mdx mice would display a reduced obesity phenotype relative to wild‐type (WT) littermates. To examine this, animals were given ad libitum access to a HFD for 8 weeks, during which weight gain, food intake, and adiposity were examined. Metabolic efficiency, defined as cumulative mass gained divided by cumulative food consumed during the 8 weeks, was also determined. As hypothesized, mdx mice were protected against excessive obesity as seen by a 35% lower adiposity index (P < 0.05) relative to WT littermates. This protection could not be explained by differences in food intake (P = 0.85). However, metabolic efficiency was 13% lower (P < 0.05) in the mdx mice, suggesting that energy expenditure was greater during the dietary period. In conclusion, mdx mice were protected against obesity despite consuming a standard Westernized HFD. Future directions will explore the possible muscular mechanism(s) that may convey this protection.
REDD1 Alters the Rate of Muscle Hypertrophy following Functional Overload
Authors: Gordon Bradley Scott , Nader Gustavo , Steiner Jennifer L , Jefferson Leonard S , Kimball Scot R
Published: November 2023

Overload‐induced skeletal muscle hypertrophy is due to an accretion of muscle protein, however the events that initiate the response are poorly understood. We recently showed that expression of Regulated in Development and DNA Damage 1 (REDD1), a repressor of signaling through the mechanistic target of rapamycin in complex 1 (mTORC1), significantly reduces the rate of protein synthesis following an acute bout of high frequency muscle contractions. However, it is unknown whether REDD1 expression alters the rate of overload‐induced muscle hypertrophy. To address this question, REDD1+/+ and REDD1−/− mice were subjected to unilateral functional overload (OV) of the plantaris by removal of the synergists while the contralateral sham leg served as an internal control. Mice were allowed to recover for 3 or 5 days. After 3 days of OV, mice were fasted for 16 hr, and puromycin was injected 30 min prior to sacrifice for measurement of the rate of protein synthesis. The percent increase in plantaris wet weight relative to sham was significantly greater in REDD1−/− mice at both time points which was due to increased protein content. Further examination of the early events after 3 days of OV revealed a similar rate of protein synthesis between genotypes despite increased mTORC1 activation in the REDD1−/− mice compared to controls (phosphorylation of p70S6K1 (Thr389) and 4E‐BP1 (Ser65)). Additionally, the increase in RNA content as well as the content of the 45S pre‐rRNA was not different between genotypes after 3 days OV. There was also no difference in the content of ubiquinated proteins between genotypes. These data suggest REDD1 expression regulates muscle mass following OV due to initiating events that occur prior to 3 day of OV.

Support or Funding Information

Supported by NIH grant DK15658 (LSJ)
The role of denervation in cytokine‐mediated muscle dysfunction in muscles of old mice
Authors: Staunton Caroline Amy , Pollock Natalie , Lightfoot Adam , Vasilaki Aphrodite , Barrett‐Jolley Richard , McArdle Anne , Jackson Malcolm
Published: November 2023

Age‐related loss of muscle mass and function occurs over a substantial portion of later life and greatly influences the quality of life of older people. The causes of this dysfunction are unknown, but substantial remodelling and loss of motor units and denervation are evident in muscles of old mice. Full denervation of muscle caused elevations in the rate of mitochondrial H202 production. Data from our laboratory have demonstrated that H202 was elevated in muscle at 3 days post‐denervation and remained significantly elevated for 10 days, compared with sham controls. Further work from our laboratory has shown that this increase in H2O2 is also evident in the innervated portion of partially denervated muscle. The aim of this study was (1) to use intra‐vital imaging in Thy1‐CFP mice to determine the structure of peripheral axons and neuromuscular junctions in single muscle fibers in vivo and to simultaneously monitor H202 in the muscle fibers using AAV6‐Hyper2‐Cyto mice; (2) to examine the downstream effects of increased H2O2, including NF‐kB activation and production of pro‐inflammatory cytokines in whole muscles and in individual fibers and (3) whether these processes contribute to age‐related muscle dysfunction. Data demonstrate that the increased H2O2 in fully denervated muscle of adult mice was associated with activation of NF‐kB and increased production of pro‐inflammatory cytokines; IL‐6 (302% increase), MCP1 (324% increase) and CXCL1 (261% increase), all as early as 1 day post‐denervation (p<0.05, Students T‐Test), in a similar manner to quiescent muscles of old mice. These data suggest that denervation of individual muscle fibers in old mice results in significant elevations in H2O2 production in both denervated and innervated fibers and that this may play a role in the increased activation of NF‐kB and local production of pro‐inflammatory cytokines seen in muscles of old mice. Additional studies will explore neuromuscular transmission using electrophysiology in single fibers to determine the effects of different degrees of loss of neuromuscular junction integrity on neuromuscular transmission and the relationship of this to H202 production.

Support or Funding Information

Supported by the UK Medical Research Council.
Repeated bouts of resistance training with short interval activate Akt‐mTOR signaling, but not protein synthesis in mouse skeletal muscle
Authors: Takegaki Junya , Arihara Yuki , Ogasawara Riki , Tsutaki Arata , Nakazato Koichi , Ishii Naokata
Published: November 2023

Resistance training activates Akt‐mTOR signaling pathway, thereby stimulates the protein synthesis within skeletal muscle. However, insufficient recovery period between training bouts may lead to the accumulation of fatigue, known as “overtraining”.

Purpose

To obtain an insight into the mechanism of overtraining, changes in muscle protein synthesis and Akt‐mTOR signaling activity were investigated with changing the interval between training bouts.

Methods

Male C57BL/6J mice were randomly assigned into four groups: Control (Cont), resistance‐trained with 3 bouts at interval of 72h (72H), 24h (24H) and 8h (8H). The resistance exercise consisted of 50 repetitions of maximal isometric contraction of the tibialis anterior muscle. The muscle contraction was induced by transcutaneous electrical stimulation of deep fibular nerve under anesthesia. Muscle samples were collected 6h after the final exercise session, and kept frozen until analysis. Fifteen minutes prior to the muscle sampling, mice were injected with 0.04 μmol/g puromycin.

Results

Phosphorylation of Akt and p70S6K was observed in 24H and 8H, and that in 8H was significantly higher than in 24H. The phosphorylation of rpS6 was observed in all exercised groups, and was higher in 24H and 8H than in other groups. Puromycin‐incorporated proteins, i.e., newly synthesized proteins in ribosomes, increased in 72H, but not in 24H and 8H.

Conclusion

The present results suggest that bouts of resistance training with interval shorter than 24h do not cause an increase in protein synthesis, even though activate Akt‐mTOR signaling pathway.
TRB3 Regulates Denervation‐induced Muscle Atrophy and Insulin Resistance in Mouse Skeletal Muscle
Authors: Choi Ran Hee , Jeong Ha‐Won , McConahay Abigail , Hirshman Michael F. , Goodyear Laurie J. , Koh Ho‐Jin
Published: November 2023

Skeletal muscle atrophy, which is a loss of muscle mass, has been linked to the development of insulin resistance. Since skeletal muscle accounts for 40–50% of body mass in humans and functions in glucose disposal, it is important to prevent muscle loss to improve glucose metabolism and the quality of life. However, the mechanism(s) of muscle atrophy and its related decrease in glucose metabolism has not been fully understood. TRB3 is a pseudokinase that has been associated with insulin resistance in multiple tissues, including skeletal muscle and liver. Our hypotheses were that TRB3 mediates denervation‐induced muscle atrophy and insulin resistance. Here, we find that TRB3 protein expression was significantly increased in both 3 days (2.24‐fold) and 9 days (16.2‐fold) of denervation prior to muscle weight loss and that denervation for 9 days decreased muscle mass by 20–30% and impaired insulin‐stimulated IRS1 Y612 (23%) and Akt T308 (22%) phosphorylation. Overexpression of TRB3 in mouse tibialis anterior muscle significantly impaired insulin‐stimulated IRS1 Y612 (46%) and Akt T308 (23%) phosphorylation. Consistent with these findings, TRB3KO mice fed a high‐fat diet for 8 weeks exerted improved insulin signaling, including IRS1 Y612 (30%), Akt T308 (87%), FoxO1 S256 (17%), and FoxO3a S253 (41%), compared to wild type controls. Furthermore, 12‐week‐old TRB3KO mice showed a significant increase in muscle mass compared to wild type (TA; 10%, Gas; 15%) that was associated with decreased mRNA expression of muscle ubiquitin ligases, Atrogin 1 (41%) and MURF1 (42%). These data suggest that TRB3 plays a critical role in the regulation of skeletal muscle mass and glucose metabolism.
The Use of an Adenine Diet to Induce Uremic Cardiac Hypertrophy in Mice
Authors: Adamic Emily M , Von Schulze Alex , Amat‐Fernandez Clara , Lee Harold , Daniel Dorothy , Wacker Michael J , Touchberry Chad D
Published: November 2023

Over 90% of patients with chronic kidney disease (CKD) will develop cardiac hypertrophy and approximately half of them will die from cardiovascular disease. Recently, our lab and others have shown that uremic toxins like fibroblast growth factor 23 can directly induce pathological cardiac hypertrophy. While the understanding of the mechanisms responsible for cardiac hypertrophy in uremia is improving, there is a strong need to establish a simple, non‐surgical, and reproducible mouse model of uremic cardiac hypertrophy. An adenine diet has been shown to cause nephropathy and uremia; however, the link between ingestion of adenine and uremic cardiac hypertrophy has not been established. The objective of the present study was to develop a mouse model of dietary adenine‐induced uremia that results in cardiac hypertrophy. Thirty CD1 mice were randomly divided into two groups and fed for 12 weeks: 1) a control group (CON, n=15) fed standard feed, and 2) an adenine (0.2–0.15%) diet intervention group (AD, n=15). The percentage of adenine was adjusted weekly based on temporal assessments of blood urea nitrogen (BUN) to ensure initiation and maintenance of renal injury. Throughout the protocol, the BUN in AD mice was significantly elevated (~80 mg/dl) compared to that of the controls (~30 mg/dl). Post‐intervention, serum levels of BUN (+118%), creatinine (+72%), and phosphate (+107%) were significantly higher in the AD mice. Kidney mass/body mass ratio was significantly lower (−38%) in AD mice, suggesting renal atrophy. The AD mice also had increased heart mass/body mass ratio (+25%) and increased ventricular wall thickness, consistent with hypertrophy. Our data suggests that over 12 weeks, the induction of uremia in the AD group resulted in cardiac hypertrophy. Adenine diet fed mice may be a suitable model to study novel therapeutic treatments to prevent or treat the development of cardiac hypertrophy during uremia.

Support or Funding Information

University of Memphis Faculty Research Grant to CDT; Honors Summer Research Fellowship, University of Memphis Helen Hardin Honors Program to EMA
Striatin expression is altered during cardiac remodeling and it regulates cardiomyocyte response to adrenergic stimuli
Authors: Alotaibi Shahd , Alsolme Ebtehal , Alsomali Rahmah , Kvietys Peter , Dzimiri Nduna , Nader Moni
Published: November 2023

Mounting evidence implicates striatin in regulating cardiac function by virtue of its interaction with regulators of the L‐type calcium channel (LCC), i.e. caveolin‐3, protein phosphatase 2A, and calmodulin. We have previously shown that knockdown (KD) of striatin using shRNA (shRNA‐STRN) in neonatal rat cardiomyocytes reduced the rate of their spontaneous contraction and desensitized them to isoproterenol. We aimed to explore if the expression of STRN is altered during cardiac remodeling using Spontaneously Hypertensive Rats (SHR) and if STRN modulates the adrenergic response in cardiomyocytes. Herein, we show that STRN expression is higher in the left ventricle (LV) of SHR when compared to Wistar Kyoto (WKY) rats at the age of 4 weeks. Surprisingly, at the age of 18 weeks, the expression of STRN in LV was less in SHR when compared to WKY rats. KD of STRN in cultured cardiomyocytes did not affect the expression of the LCC. However, confocal microscopy revealed that an isoproterenol challenge resulted in less intracellular calcium [Ca2+]i in cardiomyocytes with lower amounts of STRN (KD) when compared to control cells (benign shRNA). Moreover, the rise in [Ca2+]i with respect to the baseline levels, was lower in shRNA‐STRN cardiomyocytes. Collectively, our data show that STRN levels are altered during the development of cardiac pathology. In addition, STRN plays a fundamental role in regulating cardiac [Ca2+]i responses to adrenergic stimuli and emerges as a novel therapeutic target for cardiac diseases.

Support or Funding Information

This work is supported by a KACST operating grant (#13‐BIO1452‐20) to MN and PK, and by Alfaisal University.
High‐Throughput Contractile Force Screening for Discovery of Novel Bronchodilators
Authors: Park Chan , Burger Stephanie , Watts Eleanor , Frykenberg Matthew , Tambe Dhananjay , Zhou Enhua , Krishinan Ramaswamy , Marinkovic Aleksander , Tschumperlin Daniel , Butler James , Solway Julian , Fredberg Jeffrey
Published: November 2023

In pathologies of the heart, blood vessels, and airways, and in cancer as well, the ultimate therapeutic target is often modulation of cellular contractile force. For discovering new therapies, drug screening technologies typically use biochemical or structural surrogates for contractile force rather than contractile force itself. Because such approaches are indirect, some hits are false while other potential hits could be missed. To fill this gap, we developed a new high‐throughput method that utilizes cellular contractile force directly as the reporter ‐‐called contractile force screening (CFS)‐‐ and established feasibility of CFS in the context of drug discovery in asthma. During an acute asthma attack, contractile forces generated by airway smooth muscle cells act to constrict the airway and thus obstruct airflow. To dilate constricted airways, asthma patients use bronchodilators to reduce these contractile forces and thus allow the airway to open more fully, but currently available bronchodilator medications often fail to relax that muscle sufficiently, especially in severe asthma. To address this unmet need for more effective bronchodilator medications, we implemented CFS to evaluate 10,000 small molecule compounds selected randomly from the Chembridge DIVERset® library.

Acrylamide gels were made in 96‐well plates and fluorescent markers and collagen were coated onto gel surfaces. Upon each gel we cultured primary human airway smooth muscle (HASM) cells to near confluence. Drug effects were quantified as the ‘force response ratio’, namely, the ratio of the contractile forces before and after drug addition. By screening mixtures of 8 compounds simultaneously, we were able to evaluate 2000 compounds per week in quadruplicate. After retesting individual compounds from initially positives, we identified 2 individual compounds that relaxed HASM cells in a dose‐dependent and a non‐toxic manner. The mechanism of action is still under investigation but we found that these compounds work independent of cAMP. CFS provides a convenient, rapid, and inexpensive platform for drug discovery based upon a physiological endpoint, namely, cell contractile force. Furthermore, CFS technology has the potential to become a new high‐content assay for drug discovery in any disease process in which cellular contractile force plays a central role.

Support or Funding Information

NIH P50HL107171 (CADET phase I), R01EY019696 (Eye BRP), NRF GRN (NRF‐2013S1A2A2035518)
Ablation of the Semaphorin 3F‐Neuropilin 2 Axis Increases Contractility in Bladder Smooth Muscle
Authors: Bielenberg Diane R , Vasquez Evalynn , Doyle Claire , Lukianov Stefan , Cristofaro Vivian , Sullivan Maryrose P , Adam Rosalyn M
Published: November 2023

Introduction

Impairment of the strength or duration of bladder smooth muscle (SM) contraction characterizes a prevalent but poorly understood clinical condition termed detrusor underactivity (DU). DU is a complex disorder that arises from diverse neuromuscular insults. DU reduces voiding efficiency and can lead to significant urologic complications. Despite significant morbidity, no effective treatment exists for this condition. Neuropilins (Nrp) are transmembrane receptors that bind class 3 semaphorin (Sema3) proteins. We identified bladder SM as a major site of expression of Nrp2 in vivo, and showed that bladder SM strips from mice with genetic deletion of Nrp2 displayed increased contractility compared to those from wild‐type littermates, identifying Nrp2 as a negative regulator of SM contractility. In this study we expanded our analysis of this signaling axis by examining the localization of the Nrp2 ligand, Sema3F, in bladder tissue and compared the contractility of bladder SM in Sema3F knockout mice compared to wild‐type mice. We tested the hypothesis that Nrp2 deletion in vivo improves bladder SM function under conditions of DU.

Methods

Nrp2 and Sema3F protein expression was localized by immunohistochemistry. Partial bladder outlet obstruction (pBOO) was created surgically in male mice at 3 mos of age by urethral occlusion. We used SM22a‐CreERT2:Nrp2fl/fl mice to achieve tamoxifen‐inducible SM‐specific Nrp2 knockout either before creation of pBOO or during the decompensation phase following pBOO. Nrp2‐expressing mice (Nrp2fl/fl mice) were used as controls and sham surgery was performed to control for pBOO. Bladder SM contractility was assessed by isometric tension testing.

Results

Sema3F expression was found in the urothelium and Nrp2 expression was highest in the bladder SM, suggesting a paracrine signaling axis. Sema3F deletion resulted in increased contractility of bladder SM similar to that seen in Nrp2 KO mice. After pBOO, bladder weight increased in both Nrp2‐intact and Nrp2‐deleted mice. Bladder SM from mice in which Nrp2 was deleted prior to pBOO displayed increased contractility at all time points after pBOO compared to tissues from Nrp2‐intact mice. Bladder SM from mice in which Nrp2 was deleted during decompensation showed increased force of contraction compared to obstructed mice in which Nrp2 was not deleted (p<0.05).

Conclusions

These observations demonstrate that downregulation of Nrp2 by genetic deletion promotes increased bladder SM contractility under conditions of chronic bladder outlet obstruction. Importantly, they show that deletion of Nrp2 in mice that have already undergone bladder decompensation can restore SM contractile function, suggesting that Nrp2 may be a therapeutic target in conditions characterized by detrusor underactivity.

Support or Funding Information

Vascular Biology Program, Boston Children's Hospital; NIDDK DK65298
Integrin‐Linked Kinase Enhances Skeletal Muscle Fatty Acid Metabolism In Vivo
Authors: Lark Daniel S , Trefts Elijah , James Merrygay , Zent Roy , Pozzi Ambra , Wasserman David H
Published: November 2023

Accumulation of extracellular matrix (ECM) components and activation of specific integrin receptors contribute to skeletal muscle (SkM) insulin resistance in the high fat (HF)‐fed mouse. Integrin‐linked kinase (ILK) is an adaptor protein that serves as a hub for the integrin signaling but its role in metabolism is poorly defined. A shift from glucose to fatty acid (FA) oxidation is a hallmark of insulin resistance and diabetes, but the mechanisms responsible for this shift in nutrient utilization are unclear. Integrins have been implicated in the transcriptional regulation of mitochondrial biogenesis and are known to interact with FA transporters at the plasma membrane. The objective of this study was to test the novel hypothesis that integrins, through ILK signaling, enhance SkM FA metabolism under insulin resistant conditions.

SkM‐specific ILK knockout mice (mILK‐KO) were generated using mice expressing Cre recombinase on the human SkM actin (HSA) promoter. Body weight was measured weekly and body composition was measured immediately prior to and following energy balance studies. mILK‐KO and their wild type (WT) littermates were fed a chow or HF diet (60% calories from fat) for 27 weeks then placed in metabolic cages for five days (n=6–9/group). Tissues were collected after a 5‐h fast. Pyruvate and FA‐supported mitochondrial respiratory capacity was measured in permeabilized SkM myofibers (PmFBs) from the soleus and EDL. Muscle free fatty acids (FFAs) and triglycerides (TGs) were measured in gastrocnemius with gas chromatography. Comparisons were made using Student's t‐test or 2‐way ANOVA as appropriate.

HF‐fed mILK‐KO mice had a decreased reliance on FA oxidation compared to their WT littermates, as suggested by the increase in RQ. This RQ difference was independent of changes in whole body energy expenditure, body composition, food consumption or spontaneous activity. RQ was unchanged in mILK‐KO mice fed a chow diet. FA‐supported PmFB respiration was decreased in EDL, but not soleus, of HF‐fed mILK‐KO mice. Respiration in chow‐fed mice was not affected by muscle ILK deletion. Total muscle FFAs and TGs were increased by HF feeding but lower in mILK‐KO mice fed a HF diet. Muscle lipids were unaffected by ILK deletion in mice on a chow diet.

These data are the first to demonstrate a mechanistic role for integrin signaling in the regulation of SkM FA metabolism in vivo and suggest that integrin signaling, through ILK, responds to nutrient overload by enhancing the uptake, storage and oxidation of FAs in SkM. These findings provide strong support for a model where extracellular cues (e.g. ECM expansion) cause a coordinated shift in cellular energetics, a paradigm that may permit for therapeutic targeting of oxidative metabolism through integrin receptors.

Support or Funding Information

Funding for this project was provided by the following NIH grants: R01 DK054902, U24 DK059637, P30 DK020593, and T32 DK007061.
Exercise Mitigates Cognitive Functions through Mitochondrial Remodeling in Type‐2 Diabetes
Authors: Kalani Anuradha , Chaturvedi Pankaj , Winchester Lee J , Tyagi Suresh C , Tyagi Neetu
Published: November 2023

Abnormal brain mitochondrial functions result in cognitive impairments during type‐2 diabetes (T2D). Exercise has been recommended by clinicians to diabetics as a secondary protective therapy; however, its effect on alleviating brain functions through mitochondrial interventions is not much explored. Therefore, we hypothesize that exercise mitigates T2D‐induced neurodegeneration and cognitive functions through mitochondrial remodeling. To test this hypothesis, we used obese diabetic db/db (leptin receptor knockout) and db/+ mice as controls, with and without exercise. The mice were exercised at a constant speed of 7 meters/min for 5 days a week/total 8 weeks. Diabetic mice with exercise had reduced total body weight (R2=0.9154, F=72.15, p<0.001), blood glucose (R2= 0.9264, F=83.92, p<0.001), liver weight (R2=0.9703, F=217.6, p<0.001) and improved heart weight (R2=0.7572, F=20.79, p<0.001) as compared to other experimental groups. However, no significant differences were observed in the weight of kidney, brain, and lungs. We next assessed cognitive and behavior alterations by open field, novel object recognition, Y‐maze, and hot plate analgesia tests. Our results suggest significant improvement in behavior and cognitive functions after exercising diabetic db/db mice as compared to non‐exercised db/db and control littermates. Results with RT2 profiler PCR array, designed especially for mitochondrial genes, confirmed improvements in mitochondrial transport, dynamics, and regulatory genes in exercised db/db brain as compared to non‐exercised db/db brains. The involvement of mitochondrial remodeling in db/db mice following exercise was further confirmed by significant increase in ATP production, membrane potential, copy number as well as decrease in reactive oxygen species when compared with non‐exercised db/db and control mice. Furthermore, considerable reduction in TUNEL reactivity was determined in intact mitochondria isolated from exercised db/db mice as compared to non‐exercised and control mice. In addition, the effect of exercise on neuronal survival in diabetic brain was confirmed by decrease in fluoro‐jade C reactivity that particularly stains degenerating neurons. Taken together, our results indicate myriad beneficiary effects of exercise over metabolic and mitochondrial pathology in diabetes. Furthermore, our findings suggest regular exercise mitigate neurodegeneration and cognitive disorders thereby improving total mitochondrial health.

Support or Funding Information

This work was supported by NIH grant HL107640‐NT.
Defective autophagy causes a maladaptive cardiac phenotype to exercise that leads to premature death and FGF21‐mediated protection against obesity and insulin resistance
Authors: Lira Vitor A. , Kronemberger Ana , Call Jarrod A. , Caster Hannah M , Pereira Renata O. , Laker Rhianna C. , Zhang Mei , Yan Zhen
Published: November 2023

Autophagy is induced by exercise in skeletal and cardiac muscle. Skeletal muscle autophagy is required for exercise‐induced metabolic adaptations; yet, impaired skeletal muscle autophagy may cause a compensatory ER stress response that protects against diet‐induced obesity and insulin resistance. The functional importance of cardiac autophagy to the benefits of exercise is uncertain. Here, we generated heart & muscle‐specific Atg7 knockout (Atg7h&mKO) mice to gain further insight into this apparent paradox related to skeletal muscle autophagy and to define the role of cardiac autophagy in exercise‐induced protection against obesity and insulin resistance. Despite exhibiting minimal autophagy in skeletal muscle and heart, Atg7h&mKO mice presented normal exercise capacity and glucose tolerance with a slight reduction in body weight (~5%) at 12–14 weeks of age. After 12 weeks on high‐fat diet (60% calories from fat) sedentary Atg7h&mKO mice gained weight and developed insulin resistance like control littermates (Atg7fl/fl). Exercise (voluntary wheel running) reduced the degree of diet‐induced obesity and glucose intolerance in Atg7h&mKO mice more efficiently; but surprisingly led to premature death of ~40% of these animals. This reduced survival was likely caused by a maladaptive cardiac phenotype as exercise induced physiological cardiac hypertrophy and prevented diet‐induced fibrosis in the hearts of Atg7fl/fl, whereas it exacerbated fibrosis and caused pathological hypertrophy in the hearts of Atg7h&mKO. Interestingly, this maladaptive cardiac phenotype to exercise was accompanied by improved skeletal muscle insulin sensitivity, increased Glut4 mRNA in adipose tissues, and elevated Pgc‐1a and Ucp1 mRNA in brown adipose tissue of Atg7h&mKO suggesting a potential cross‐talk between the heart and peripheral tissues. Analysis of serum indicated a robust increase in circulating FGF21 levels in Atg7h&mKO that exercised, which were accompanied by marked upregulation of Fgf21 mRNA expression exclusively in the heart. Examination of β‐Klotho mRNA, a co‐receptor required for FGF21 signaling, revealed a ~2‐fold upregulation exclusively in white and brown adipose tissues. Our results indicate that autophagy plays a critical role in exercise‐induced protection against cardiac dysfunction in obesity and diabetes. Impaired cardiac autophagy causes a maladaptive phenotype to exercise that leads to premature death, but also potentiates the peripheral metabolic benefits of exercise, at least in part, via a heart‐adipose tissue signaling axis involving FGF21.

Support or Funding Information

Obesity Research and Education Initiative (OREI) and the Fraternal Order of Eagles Diabetes Research Center (FOEDRC) at the University of Iowa (V.A.L.) and U.S. National Institutes of Health (NIH) grant AR050429 (Z.Y.)
Air puffs as refinement of electric shocks for stimulation during treadmill exercise test
Authors: Lamy Sébastien , Cartoni Cristina , Bichat Arnaud , Combe Roy , Doenlen Raphael , Warot Xavier
Published: November 2023

Endurance treadmill test is a convenient method for easily and systematically evaluate exercise performance and capacity in mice. Although mice are naturally prone to run, an aversive stimulation is required to force all animals to stay engaged in the test. This is typically achieved using mild electrical stimulation on a dedicated grid at the rear of the running belt. Another system replacing the electric stimulation by air puff was newly developed. This system seems to be an ethical evolution in regards of the 3R's principles since the electrical aversive stimulus is replaced by a less noxious stimulus. Exhaustive endurance protocol was used to compare the two different treadmill systems. This test consists to expose mice to a long lasting middle intensity exercise protocol until they reach exhaustion. In addition of the exercise performance readouts, the adaptive mechanisms established to supply fuel to skeletal muscle during exercise and stress were respectively assessed by the analysis of biochemical parameters and markers of stress produced by the hypothalamic‐pituitary‐adrenal axis. The results obtained for the exhaustive protocol are comparable in terms of behavioral and physiological parameters (e.g. running distance, running time and physiological decrease of triglycerides and hepatic glycogen level). Interestingly, corticosterone and adrenocorticotropic hormone (ACTH) levels were similar between the two types of stimulation. These results demonstrate that air puff stimulation could be an interesting alternative for the replacement of the electric stimulation during an exhaustion protocol. However, the stress level does not seem to be reduced when using air puff stimulation.

Support or Funding Information

Ecole Polytechnique Fédérale de Lausanne, School of Life Sciences, Center of PhenoGenomics
3D Virtual Histology of Trabecular and Cortical Architecture in Living Human Vertebrae
Authors: TANIOKA HISAYA , KAGA KIMITAKA
Published: November 2023

Purpose

Toprovide a non‐large‐scaled device but a small‐sized device. Less‐expensive and simple device that can be utilized for making 3D trabecular and cortical structure of living human vertebrae, and to learn about the bone strength from trabecular structure obtained by this created method.

Subjects and Methods

This study involved 15 patients who underwent body CT exams for their diagnosis. Scanning of the body was performed using collimation and pitch values of 1.00 mm and 1.00 respectively with bone algorithm. The field of view was 25.0 cm using a 512 matrix. After transferring the image data to a CT work station, 3D visualization based on interactive direct volume rendering was made using the current distributed software. The defined threshold CT values of this image processing were following; maximum value is 450 HU and minimum value is 100 HU.

Results

3D virtual histological image of the vertebra was shown in Figure. Morphometric parameters as trabecular number (Tb.N.), trabecular separation (Tb.Sp.), and trabecular thickness (Tb.Th.) were frequently used and their measured values were as followed; TbN=1.00 ± 0.10, TbSp=0.57 ± 0.07 mm, and TbTh=0.45 ± 0.03mm. These values showed significant correlations with the properties of the vertebral bone in the multiple studies.

Conclusion

This 3D virtual histological method is very simple. We can get topology and morphological parameters of living vertebrae under regular CT exams. The data of morphometric parameters suggest that this method has potential to characterize the structured implications of metabolic bone.

Support or Funding Information

Competing interests: none Sponsorship: none Funding source: none
The activation on serotonin 2B receptor (5‐HT2BR) is essential for osteoclastogenesis
Authors: Huang Yi‐Chieh , Lin Shiuan‐Yea , Lu Tzong‐Shi , Huang Bin , Lin Chih‐Lung , Fu Yaw‐Syan
Published: November 2023

Osteoporosis is the common diseases in elderly people, especially menopausal women. Excessive osteoclastogenesis is one of the main reasons to cause osteoporosis. Recent clinical researches indicated that the patients’ long‐term use of Selective Serotonin Reuptake Inhibitor (SSRI) as anti‐depression drugs usually with higher risk to suffer osteoporosis, and that indicated serotonin (5‐HT) may involve in the regulation of bone metabolism. Some previous reports showed that different types of 5‐HT receptors are expressed in both osteoblasts and osteoclasts. This study was focus on the effects of 5‐HT2BR in osteoclastogenesis. Using RANKL induced osteoclast formation by RAW 264.7 cells as the model, our data showed a positive correlation between the osteoclastogenesis with 5‐HT and 5‐HT2BR agonist treatment and that decrease SirT1 expression. Conversely, treatment with specific 5‐HT2BR antagonist significantly reduced the osteoclast numbers and increase SirT1 expression, and treatment with SirT1 agonist significantly reduced the osteoclasts. In conclusion, our study suggested that 5‐HT2BR play an essential role in osteoclastogenesis and SirT1 may be involved in regulating osteoporosis. Our observations might also provide another direction of prophylaxis and therapy for osteoporosis.

Support or Funding Information

KMU‐O105001, KMU‐TP104E34, KMU‐TP104E13
Heat Shock Protein 72 Regulates Mitochondrial Integrity and Function in the Prevention of Hepatic Insulin Resistance
Authors: Rogers Robert S. , Wheatley Joshua L. , Archer Ashley E. , Thyfault John P. , Geiger Paige C.
Published: November 2023

Nonalcoholic Fatty Liver Disease (NAFLD) encompasses a spectrum of disorders characterized by an accumulation of lipids in the liver, and is a significant risk factor for the development of insulin resistance and type 2 diabetes. Heat shock proteins (HSPs) can effectively prevent high fat diet‐induced glucose intolerance and skeletal muscle insulin resistance, improve oxidative capacity and increase mitochondrial function. The purpose of the current investigation was to determine whether HSP72, in particular, is protective against the development of hepatic triglyceride accumulation and insulin resistance. Male Wistar rats were fed a high‐fat diet (HFD) and received either weekly heat treatment (41°C for 20 min) or sham treatment (37°C for 20 min). In addition, primary hepatocytes were isolated from male C57BL/6 mice and subjected to either a single heat (42°C for 30 min) or sham (37°C for 30 min) treatment, transfected with HSP72 siRNA or exposed to insulin (60 μU/ml). Confocal microscopy images were obtained from cells incubated with tetramethylrhodamine (TMRE) and MitoTracker Green to assess mitochondria functionality. Weekly in vivo heat treatments reduced liver triglyceride accumulation, increased HSP72 levels, and modulated the protein levels of microtubule‐associated protein light chain 3 (LC3‐II) and p62 – two markers of autophagy. HSP72 siRNA in primary hepatocytes resulted in reduced mitochondrial function, reduced phosphorylation of AMP‐activated protein kinase (AMPK) and Akt, increased Parkin levels, and decreased LC3‐II levels. Our results indicate that weekly heat treatments protect against liver triglyceride accumulation in rats, and data from primary hepatocytes suggest a critical role for HSP72 in maintaining hepatic mitochondrial integrity and function. Our findings identify potential new HSP72‐mediated therapeutic targets for the regulation of hepatic mitophagy and prevention of metabolic disease.

Support or Funding Information

This work was supported by National Institutes of Health Grants AG‐031575, DK‐088940, the University of Kansas Medical Center Biomedical Research Training Program, and core support was provided by National Institute of Child Health and Human Development Grant HD‐002528.
Altered metabolism in RGS2 KO mice
Authors: Lee Katherine , Gros Robert , Chidiac Peter
Published: November 2023

Regulator of G‐protein signaling 2 (RGS2) is known to inhibit Gq‐ and Gs‐mediated GPCR signalling. We have previously reported that RGS2 knock out mice (rgs2−/−) exhibit a lean phenotype compared to their wildtype (WT) counterparts, with significantly reduced fat deposits, leptin levels, and serum lipids. The objective of this study is to further elucidate the role of RGS2 in metabolism. CLAMS metabolic cages were used to measure various indices in 1 month‐, 3 month‐, and 12 month‐old rgs2−/− and WT mice. Animals were acclimatised in metabolic cages for 24 hours (12‐hour light and dark cycles), followed by another 24 hours of metabolic recordings.

At both 3 months and 12 months, rgs2−/− mice displayed increased rates of respiratory intake (vO2) and expiratory output (vCO2), particularly during the dark cycle, suggesting increased metabolic activity in the knockouts. Furthermore, calculated respiratory exchange ratios (RER) suggest an increase in carbohydrate rather than fat or protein metabolism in rgs2−/− mice of all three age groups compared to WT. Hyperphagia, as well as increased water intake, were also observed in the knockouts at 3 and 12 months. Even with significantly increased food and water intake, 3 and 12 month‐old rgs2−/− mice maintained lower body weights compared to WT, and displayed comparable ambulatory activity.

In addition, a 30% diet restriction study in which 3 month‐old mice were fed once daily, followed by metabolic cage measurements, was performed to determine possible alterations in metabolism following changes in food intake. Compared to our previous ad libitum studies, the difference in vO2 and vCO2 between WT and rgs2−/− mice was further exacerbated. In addition, RERs were significantly higher in rgs2−/− mice, and allude to the possibility of elevated carbohydrate metabolism in rgs2−/− mice. Ambulatory activity remained comparable to WT. Interestingly, diet restriction appears to have exacerbated the hyperphagic phenotype of rgs2−/−mice – although body weights remained stable, rgs2−/−mice consumed 50% of their daily food amounts at a 4 times higher rate than WT, and displayed hoarding behaviour.

The lack of changes in overall activity between genotypes in both ad libitum and diet restricted mice suggests that weight loss in rgs2−/− mice is due to internal energy metabolism rather than overt physical activity. These results suggest that RGS2 may play an important role in the regulation of body metabolism.

Support or Funding Information

Canadian Institutes of Health Research, Heart and Stroke Foundation of Ontario
Effect of canary grass (Phalaris canariensis) seed extract on the lipid profile of rats with induced hyperlipidemia
Authors: Vizconde ‐ Rodríguez Ana Paula , Salazar ‐ Castillo Marco Leoncio , Rodríguez ‐ Haro Icela Marisa , Calderón ‐ Peña Abhel Arthur , Fernández‐Rodríguez Lissett Jeanette
Published: November 2023

Hyperlipidemia is a major risk factor in cardiovascular diseases; however, the consumption of certain plants favorably affects blood lipid profile. The aim of this study was to determinate the effects of the hydroalcoholic extract of canary grass seeds at 0.1 mg/kg and 0.2 mg/kg concentration on the lipid profile of rats with induced hyperlipidemia. The rats were randomly divided into five groups as follows: a target group, a control group, atorvastatin group and two treatment groups. There was no significant difference observed between the treatment groups.
The Effect of JAK‐Inhibitor Tofacitinib on Intestinal Epithelial Barrier Function
Authors: Sayoc Anica , Krishnan Moorthy , McCole Declan F.
Published: November 2023

Tofacitinib is an FDA‐approved oral Janus kinase (JAK) inhibitor that modulates the JAK‐STAT signaling cascade activated by inflammatory cytokines, and has shown clinical benefit in trials on ulcerative colitis (UC) patients. However, the effect of this drug on intestinal epithelial cells (IEC) and intestinal permeability, which is increased in UC, has not been elucidated. The aim of this study was to determine if tofacitinib can reduce IEC permeability caused by the inflammatory cytokine, IFN‐γ

Methods

T84 intestinal epithelial cell lines were cultured on transwells for 12–13 days. Preventive effects of tofacitinib were determined by pre‐treatment with tofacitinib (50μM) apically (to mimic oral administration to patients) for 1 hour followed by basolateral administration of IFN‐γ (1000U/ml) for 24 hours. The concentration of tofacitinib was based on a dose response (2μM, 10μM, 50μM) study on transepithelial electrical resistance (TEER) upon IFN‐γ challenge. Therapeutic effects of tofacitinib were studied by treating T84 cells twice with tofacitinib (16.7μM) at 6 and 18 hours post‐IFN‐γ treatment (1000U/ml). Timing and dosage of tofacitinib were chosen from post‐IFN‐γ dose‐response studies. TEER and 4kD FITC‐dextran permeability were measured to indicate changes in paracellular permeability to ion flux and macromolecules, respectively.

Results

Tofacitinib prevented IFN‐γ‐induced increases in TEER (p<0.001; n=3) and 4kD FITC‐dextran permeability in IECs and maintained the barrier integrity at levels comparable to untreated and vehicle (DMSO)‐treated controls. Although one dose of tofacitinib 24 hours post‐IFN‐γ had no effect on TEER, administration of tofacitinib at two time points after IFN‐γ partially rescued IECs from the effect of IFN‐γ on TEER and 4kD FITC‐dextran permeability compared to untreated and vehicle‐treated controls. These data indicate that tofacitinib can restrict intestinal epithelial barrier defects induced by an inflammatory cytokine. This may potentially contribute to the beneficial effects of tofacitinib observed in clinical trials in UC patients.

Support or Funding Information

Supported by NIH R01 DK091281 (DFM)
Intestinal Epithelial IL‐15 Overexpression Enhances γδ T Cell Mucosal Surveillance
Authors: Edelblum Karen L , Jabri Bana , Turner Jerrold R
Published: November 2023

Intraepithelial lymphocytes (IEL) expressing the γδ T cell receptor bridge innate and adaptive immunity through their high level of constitutive activation and surveillance‐like migratory behavior. Although epithelial IL‐15 is critical for the development, proliferation and survival of γδ IELs, the role of IL‐15 in γδ IEL migration remains unclear. Transpresentation of membrane bound IL‐15 requires cell‐cell contact between activating and responder cells, thus we tested the hypothesis that compartmentalized IL‐15 overexpression within the intestinal mucosa directly affects γδ IEL localization. To this end, GFP γδ T cell reporter mice were crossed to transgenic (Tg) mice overexpressing murine IL‐15 either in the intestinal epithelium (vil‐IL‐15) or lamina propria via a MHC class‐I restricted promoter (Dd‐IL‐15). Consistent with the role of epithelial IL‐15 in IEL proliferation, vil‐IL‐15 Tg mice exhibited a 61% increase in γδ T cell number compared to wildtype (WT). Time‐lapse video microscopy revealed enhanced γδ IEL migration into the epithelium in vil‐IL‐15 Tg mice relative to WT (44±8.0% vs 32±3.3%) leading to increased γδ IEL/epithelial interaction (4.5±0.2 vs 3.5±0.2 interactions/hr). In contrast, the frequency of γδ IELs within the intraepithelial compartment (25±6.8%) is reduced in Dd‐IL‐15 Tg mice, resulting in increased γδ T cell localization to the lamina propria. Notably, more epithelial apoptosis was observed in Dd‐IL‐15 Tg mice than in WT. These data indicate that epithelial IL‐15 production plays an important role in maintaining both γδ IEL number and epithelial surveillance, whereas sustained γδ T cell recruitment to the lamina propria may adversely affect epithelial survival.

Support or Funding Information

Funding: NIH, RBHS Chancellor Scholar Award