bluuebunny/crossref_metadata_2025 huggingface dataset:
K (Lysine) Acetyltransferase 6B (Kat6B) is Upregulated during Neurogenic Skeletal Muscle Atrophy and is Differentially Expressed in MuRF1‐null Mice
Authors: West Rita , Waddell David
Published: November 2023

Skeletal muscle atrophy is characterized by a decrease in muscle size and strength and results from a range of physiological conditions, including denervation, corticosteroid exposure and aging. The K (lysine) acetyltransferase 6B (Kat6B) gene, also known as Myst4, has been identified as a novel gene in skeletal muscle and was found to be differentially regulated in wild‐type mice compared to Muscle RING Finger 1 (MuRF1) knockout mice in response to denervation. Muscle RING Finger 1 (MuRF1), an E3 ubiquitin ligase, has previously been shown to be upregulated during almost all conditions of muscle wasting and is believed to play an important role in targeting proteins for degradation. However, preliminary data described in this study provides new evidence that MuRF1 may also act as a transcriptional modulator of atrophy‐induced gene activity, including the regulation of Kat6B. The Kat6B gene currently has two known and validated isoforms and 8 additional putative isoforms in mice. Microarray data revealed that Kat6B expression is significantly induced at 3 days and 14 days post‐denervation in wild‐type mice, while Kat6B expression is dramatically blunted in MuRF1‐null mice at both 3 days and 14 days post‐denervation. The Kat6B gene codes for a histone acetyltransferase that is believed to be part of the MOZ/MORF protein complex and is thought to be involved in transcriptional regulation. Interestingly, Kat6B has been shown to be required for Runt‐related transcription factor 2 (Runx2) dependent transcriptional activation. Runx2 codes for a transcription factor that plays an important role in bone and cartilage development and is closely related to Runx1. Runx1 is significantly upregulated in response to neurogenic skeletal muscle atrophy and has been shown to be required to sustain muscle mass and integrity following an atrophy‐inducing event. The results presented here in combination with additional research into the transcriptional regulation and function of Kat6B will help contribute new insight into the molecular genetic mechanisms of skeletal muscle atrophy and could provide a more complete understanding of how MuRF1 may transcriptionally regulate neurogenic atrophy‐induced gene expression during muscle wasting.

Support or Funding Information

University of North Florida Transformational Learning Opportunity Grant
Mouse Cachectic Skeletal Muscle mTORC1 Signaling in Response to Acute Eccentric Contractions
Authors: Hardee Justin P , Gao Song , VanderVeen Brandon N , Fix Dennis K , Hetzler Kimbell L , Carson James A
Published: November 2023

Eccentric contraction (EC) induced skeletal muscle hypertrophy involves the activation of protein synthesis through mTORC1 signaling. Cancer cachexia disruption of muscle protein turnover regulation is well established, but whether increased use regulates suppressed muscle protein synthesis warrants further investigation. We recently demonstrated severely cachectic muscle can initiate a growth response to repeated ECs. However, the EC activation of mTORC1 signaling in cachectic muscle has not been investigated. The purpose of this study was to determine if mTORC1 signaling and protein synthesis in cachectic muscle from 2 distinct preclinical cancer cachexia models could respond to EC. Male, ApcMin/+ (N=5; 15% body weight loss) and Lewis Lung Carcinoma (LLC) tumor‐bearing (N=8; 14% body weight loss) mice performed 1 bout (10 sets of 6 repetitions) of EC and were sacrificed 3h post‐contraction. The left TA performed EC while the right TA served as intra‐animal control. C57BL/6 (WT) mice served as controls. In control muscle ApcMin/+ and LLC mice had decreased muscle mass, p70S6K(T389) phosphorylation, and protein synthesis compared to WT controls. Cachexia did not inhibit the acute EC‐induction of p70S6K(T389) phosphorylation in ApcMin/+ or LLC mice. While EC induced protein synthesis in ApcMin/+ mice, protein synthesis remained suppressed compared to WT mice. Interestingly, EC did not stimulate muscle protein synthesis in LLC mice. Although cachexia suppressed muscle anabolic signaling, cachectic muscle maintains the ability to activate mTORC1 signaling by EC. However, in LLC‐induced cachexia there is a disconnect between mTORC1 activation and the induction of protein synthesis. Further studies are required to determine the tumor specific effects that regulate the anabolic response to eccentric contractions in cachectic muscle.

Support or Funding Information

Supported by NIH/NCI R01‐CA121249
Proteotyping For Drug Development: Proteomics Impacting Clinical Practice
Authors: Van Eyk Jennifer , Holewinski Ronald , Wang Shengbing , Kirk jonathan , Kass David
Published: November 2023

The road to medical translation starts with understanding of an individual's history and disease status via the quantification of proteins and their disease‐induced modified forms. Each individual's proteome reflects both their cellular memory and current health status and should allow prediction of their response to environment and therapeutic treatment. Thus, precision medicine maybe feasible but it will require the ability of specific drug targeting at the level of an individual. One example is the targeting of drugs to activate the protein kinase G (PKG) pathway, which in animal studies have been shown to blunt the development of heart failure. Drugs that inhibit the breakdown of cGMP (thus, activating PKG) have had less than expected therapeutic efficacy in numerous clinical trials. We have recently shown that there are multiple unanticipated levels of regulation of the PKG pathway that influence which subproteome is affected by PKG that ultimately leads to varied functional consequences. This includes an individuals’ redox status and heart failure type (e.g heart failure with preserved ejection fraction) that should affect the effectiveness of the current PKG‐targeted drugs. Similarly, resynchronization therapy, one of the few clinically used treatments for heart failure where dual pacemakers are implanted to ensure coordinated cardiac pumping, activates GSK 3 beta altering the contractile subproteome. This kinase is also influenced by the redox status and the oxidation of GSK 3 beta changes its downstream targets and potentially the functional outcome. Methods to assess individual's status prior to treatment selection will increase efficacy and patient selection as well alteration to their basal/pretreatment status could increase an individual's response to therapy.

Support or Funding Information

P01 HL77189‐01, R01HL119012, NHLBI‐HV‐10‐05 (2)
Downregulation of myocardial pigment epithelium‐derived factor in mice exposed to cigarette smoke: A possible mechanism for smoking‐related cardiovascular disease
Authors: McHowat Jane , Kuenzel Hannah , Kispert Shannon , Marentette John , Kolar Grant
Published: November 2023

Cigarette smoking is estimated to cause 10% of cardiovascular disease with approximately 6 million deaths per year worldwide. Despite the well‐established correlation between smoking and cardiovascular disease, many fundamental questions regarding underlying mechanisms remain unanswered. We have shown that smoking is associated with platelet‐activating factor (PAF) accumulation, an inflammatory metabolite that is implicated in ischemia/reperfusion injury and heart failure progression. PAF enhances matrix metalloproteinase (MMP) activation, which may contribute to downregulation of pigment epithelium‐derived factor (PEDF), a glycoprotein with anti‐inflammatory and antioxidative properties that can protect against endothelial damage, platelet aggregation and T –cell activation. We hypothesized that PAF accumulation and/or PEDF downregulation may directly contribute to smoking‐induced cardiovascular disease. We exposed mice to cigarette smoke for up to 6 months and measured biochemical and histological changes in the heart. Smoking resulted in increased calcium‐independent phospholipase A2 activity (3.46 ± 0.21 to 5.76 ± 0.64 nmol/mg protein/min, p<0.01, n=4) and decreased PAF acetylhydrolase activity (2.65 + 0.06 to 1.48 + 0.02 nmol/mg protein/min), changes that indicate increased PAF synthesis and decreased inactivation. Smoking was associated with increased MMP‐2 expression and downregulation of PEDF expression in the heart by immunoblot analysis. Immunohistochemical analysis demonstrated that the majority of PEDF was associated with cardiac myocytes and was reduced in animals exposed to cigarette smoke. Following exposure to cigarette smoking, cardiac vessels exhibited thickening of the vessel walls, particularly in the smooth muscle layer. Changes in the vessel wall were similar to those observed in PEDF knockout mice. These are the first data to implicate PEDF downregulation in smoking‐related cardiovascular disease.

Support or Funding Information

Saint Louis University Doisy Research Fund
Phosphorylation and Calcium Antagonistically Tune Myosin‐binding Protein C's Molecular Structure and Function
Authors: Previs Michael J , Mun Ji Young , Michalek Arthur J , Previs Samantha Beck , Gulick James , Robbins Jeffrey , Warshaw David M , Craig Roger
Published: November 2023

During each heartbeat, calcium activates the sliding of actin‐thin filaments towards the centers of myosin‐thick filaments to shorten the overall length of cardiac muscle cells. Cardiac myosin binding protein C (cMyBP‐C) tunes these interactions throughout the contractile cycle. cMyBP‐C's C terminus is strongly bound to the thick filament backbone and its N‐terminal domains extend away and transiently interact with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocities. Both mechanisms are tunable by phosphorylation of 4 serines within an extensible, intrinsically disordered region of cMyBP‐C's N‐terminus, the M‐domain. Does phosphorylation impact the structure and molecular mechanics of cMyBP‐C's N terminus to tune it's function? Atomic force spectroscopy studies of N‐terminal fragments (domains C1–C2), showed that phosphorylation reduced the M‐domain's contour length and extensibility. Rotary shadowing electron microscopy showed that M‐domain phosphorylation caused N‐terminal fragments (C0–C3) to shift from an extended, rod‐like structure to a compact conformation. Taken together, M‐domain phosphorylation and its impact on cMyBP‐C's N‐terminal structure suggest a mechanism for tuning cMyBP‐C's function in motility assays. Interestingly, we found that free calcium (0.1 mM), necessary to fully activate the thin‐filament, mitigated the structural effects of phosphorylation by increasing the free‐extensibility of the phosphorylated M‐domain and shifting the phosphorylated N‐terminal fragments back to the extended, rod‐like state, as if unphosphorylated. Functionally, even though phosphorylation reduced cMyBP‐C's ability to inhibit actin filament sliding velocity in the motility assay (24% vs. 40% inhibition), addition of calcium ablated the impact of phosphorylation, fully restoring cMyBP‐C's inhibitory capacity. We conclude that phosphorylation of MyBP‐C's M‐domain may have its greatest effect on cardiac contractility by tuning cMyBP‐C's ability to sensitize actin‐thin filaments to calcium at the low levels present during the onset of contraction. Importantly, calcium levels at the peak contraction would allow cMyBP‐C to remain a potent contractile modulator, regardless of the cMyBP‐C's phosphorylation state.

Support or Funding Information

NIH grants HL124041 and HL059408 supported these studies.
Effect of Hypertrophic Cardiomyopathy‐Associated R282W Mutation of MYBPC3 on M‐domain Phosphorylation in vitro
Authors: Giuffre Allison Mary , Lynch Thomas L. , Sadagopan Nandhini , Sadayappan Sakthivel
Published: November 2023

Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium characterized by left ventricular hypertrophy and contractile dysfunction. HCM is the most common inherited cardiac disease and is caused by mutations in sarcomeric proteins. Importantly, mutations in MYBPC3, the gene encoding cardiac myosin binding protein‐C (cMyBP‐C), account for 40% of all HCM cases. A missense mutation causing the amino acid substitution R282W within the M‐domain of cMyBP‐C has been previously discovered in HCM patients. This mutation directly alters the protein kinase A (PKA) recognition motif for phosphorylation of the human cMyBP‐C Ser‐284. Indeed, previous studies show that phosphorylation at the homologous murine Ser‐282 residue is a prerequisite for subsequent phosphorylation of the murine Ser‐273 and Ser‐302 residues. Given the importance of cMyBP‐C phosphorylation of these residues in regulating myofilament cross‐bridge kinetics and contraction, the R282W mutation may alter cMyBP‐C phosphorylation within the M‐domain. Therefore, we determined the effect of the R282W M‐domain mutation on cMyBP‐C phosphorylation status. We hypothesized that this mutation diminishes cMyBP‐C phosphorylation at human Ser‐284. To test the hypothesis, H9C2 cells were infected with adenovirus containing cDNA encoding full‐length human WT or R282W cMyBP‐C. After 48 hours of infection, cells were treated with or without isoproterenol, to activate PKA. Western blot analysis of total cell lysates from H9C2 cells expressing R282W cMyBP‐C demonstrated a significant reduction in phosphorylation of Ser‐284, compared to WT cMyBP‐C. In conclusion, these data suggest that the R282W mutation decreases cMyBP‐C phosphorylation at Ser‐284, which may contribute to contractile dysfunction in HCM.

Support or Funding Information

This research was funded by the American Physiological Society.
Essential roles of ANO1 in mouse urethral smooth muscle contraction and urinary continence
Authors: Feng Mei , Liu Donghai , Lu Ping , Bellve Karl , Lifshitz Lawrence M , Fogarty Kevin , ZhuGe Ronghua
Published: November 2023

Smooth muscle, the major cell type of urethra, generates sufficient intraurethral pressure to prevent voiding of urine from the bladder; its dysfunction contributes to stress urinary incontinence, a pathological disorder affecting up to 30% of women and causing physical discomfort and emotional distress. However, the cellular and molecular mechanisms underlying the contraction of urethral smooth muscle is not well understood. In the present study, we report that mouse urethral smooth muscle expresses type 2 ryanodine receptors, L‐type Ca2+ channels, and the Ca2+‐activated Cl− channel ANO1. In response to a load, mouse urethral strips relaxed and then developed a spontaneous tone. This tone was blocked by ryanodine receptor antagonist ryanodine, ANO1 antagonist benzbromarone, and L‐type Ca2+ channel inhibitor nifedipine; this tone was absent in the urethral strips from smooth muscle specific ANO1 deletion mice. Ryanodine receptor agonist caffeine contracted mouse urethral strips dose‐dependently. This contraction was inhibited by ryanodine, nifedipine, benzbromarone and ANO1 deletion. Caffeine increased global [Ca2+]i in isolated single urethral smooth muscle cells, and this increase in [Ca2+]i was inhibited by ryanodine, benzbromarone and nifedipine. In ANO1 deleted cells, caffeine caused less increase in [Ca2+]i compared to the wild type cells, and this rise in [Ca2+]i was resistant to benzbromarone and nifedipine. Caffeine induced Ca2+‐activated Cl− currents in wild type cells, and failed to do so in the ANO1 deletion cells. The size of urine stains was greater while the number of stains was smaller in wild type mice than in ANO1 deletion mice, implying ANO1 deletion may impair urinary continence. We conclude that ANO1 is functionally coupled with ryanodine receptors and L‐type Ca2+ channels, and this functional coupling is required for spontaneous tone and agonist‐induced contraction in urethra, and its disruption could lead to urinary incontinence.

Support or Funding Information

NIH NIDDK098586
HIF‐1α regulates a single bout of exercise‐induced hepatic expression of glucose and lipid metabolism related genes
Authors: Luo Beibei , Xiang Dao , Chen Peijie , Hu Yiping
Published: November 2023

Exercise increases the energy demand, disrupting the hepatic glucose and lipid metabolic homeostasis. A single bout of exercise could induce tissue blood flow redistribution by which the blood flow is decreased in the splanchnic circulation. The hypoxia‐inducible factor (HIF) 1α is pivotal in the transcriptional response to oxygen flux. Therefore, we hypothesize that HIF‐1α plays an important role in the regulation of hepatic metabolism related genes expression after a single bout of exercise. The noninvasive in vivo bioluminescence imaging was applied to monitor HIF‐1α expression after exercise in ROSA26 ODD‐Luc/+ mouse model (ODD‐Luc) that ubiquitously expresses a bioluminescent reporter consisting of firefly luciferase fused to a region of HIF‐1α. We found that one session of 30min swimming exercise markedly increase in vivo HIF‐1α in the gastrointestinal (GI) system and liver. Then two small molecule inhibitors were respectively intraperitoneal (i.p.) injected to the ODD‐Luc mice: DMOG (100mg/kg), which blocks prolyl hydroxylase domain (PHD) protein thus stabilizes HIF‐1α; and PX‐478 (100mg/kg), which inhibits HIF‐1α. The data was shown that the DMOG administration increased in vivo HIF‐1α in the GI system and liver; PX‐478 administration reduced the in vivo HIF‐1α expression after one session of 30min swimming. 8‐week male FVB/N mice were divided into 5 groups (n=6 per group): one session of 30min swimming (SWIM), swimming with PX‐478 i.p. injection (SWIM‐PX478), PX‐478 i.p. injection (PX478), DMOG i.p. injection (DMOG) and control (CON). Gene expression profiling experiments were performed on mouse liver cDNA samples from each group using RT2 Profiler PCR Arrays including over 240 genes related to glucose metabolism, fatty acid metabolism, and lipoprotein signaling and cholesterol metabolism. It was found that one session of exercise‐induced genes expression were significantly different (P<0.001) between the SWIM‐PX478 and SWIM mice, including G6pc, Pck1, Pdk1, Scd1, Acadm, Acox1, Ctp1, Cpt2, Crot, Hmgcs2, Srebf and Ldlr. The pattern of one session of exercise induced hepatic metabolism related genes expression was similar between the DMOG and SWIM groups. Albumin Cre mediated, hepatocyte specific deletions of HIF‐1α or PHD2 mouse models were also developed to verify the function of HIF‐1α in regulating the hepatic glucose and lipid metabolism related genes expression after one session of 30min swimming. In conclusion, these results demonstrate an important role for the HIF‐1α in the regulation of hepatic metabolism related genes expression after a single bout of exercise.

Support or Funding Information

The National Natural Science Foundation of China (31471135) and the China Postdoctoral Science Foundation funded project.
Roux‐en‐Y Gastric Bypass Surgery Enhances Contraction‐Mediated Glucose Metabolism in Primary Human Myotubes
Authors: Hinkley J. Matthew , Zou Kai , Park Sanghee , Turner Kristen , Zheng Donghai , Houmard Joseph A
Published: November 2023

Contractile activity (e.g. exercise) invokes various metabolic adaptations in skeletal muscle, including improved insulin action and substrate oxidation. However, individuals with metabolic perturbations (i.e. severely obese (BMI ≥ 40 kg/m2) and type 2 diabetics) appear to have inherent defects in the ability to respond to contractile activity. Along with significant weight loss, Roux‐en‐Y gastric bypass (RYGB) surgery elicits positive metabolic adaptations; however, it is uncertain whether this clinical intervention alters the myocellular milieu to respond to contractile activity. To examine this, skeletal muscle cells were isolated from muscle biopsies obtained from patients before and after RYGB surgery, and differentiated into myotubes. Once fully differentiated, myotubes were electrically stimulated to contract for 24 hours (2 ms bipolar pulse, 1 Hz, and 11.5 V), after which changes in metabolic function and intracellular signaling were examined. Myotubes from severely obese patients prior to RYGB surgery were unresponsive to the metabolic benefits of muscle contractile activity, as indicated by a lack of change in insulin‐stimulated glycogen synthesis (38.6% vs. 38.5% increase over basal for control vs. electrical stimulation) and basal glucose oxidation (4.5 nmol glucose/mg/hr vs. 4.6 nmol glucose/mg/hr control vs. electrical stimulation). Interestingly, myotubes derived from the same patients 1‐month post‐RYGB were responsive to muscle contraction, as indicated by an ~1.4‐fold increase in insulin‐stimulated glycogen synthesis (36.9% vs. 52.9% increase over basal for control vs. electrical stimulation) and an ~1.5‐fold increase in basal glucose oxidation (3.9 nmol glucose/mg/hr vs. 5.5 nmol glucose/mg/hr control vs. electrical stimulation). At the molecular level, while unresponsive before surgery, muscle contraction improved insulin‐stimulated phosphorylation of the Rab‐GTPase AS160 1‐month following RYGB surgery (~1.9‐fold vs. ~2.6‐fold insulin stimulation for control vs. electrical stimulation). These data indicate that RYGB surgery inherently improves the ability of skeletal muscle to respond to contractile activity, suggesting an exercise program should be immediately implemented in these patients to further improve substrate metabolism.

Support or Funding Information

NIH DK56112
Human Energy Expenditure is in Control of Cytochrome C Oxidase Subunit IV‐2
Authors: Schiffer Tomas A , Ekblom Björn , Sundqvist Michaela Lina , Lundberg Jon O , Weitzberg Eddie , Larsen Filip J
Published: November 2023

Background

The molecular mechanisms of the pronounced between subject variation in human resting metabolic rate (RMR) remains elusive. The terminal enzyme of the mitochondrial respiratory chain, Cytochrome C oxidase (COX) plays a key role in the control of mitochondrial respiration. There are a number of regulatory mechanisms controlling COX activity and proton pump efficiency. A shift of COX subunit composition may also be a part of the regulation of the enzyme. Recent in vitro studies of COX IV‐2 has implicated this isoform in the cellular response to hypoxia and oxidative stress. The aim of this study was to investigate whether COX IV‐2 is involved in the regulation of respiration in vivo and explains the pronounced individual variations in resting RMR.

Methods

Indirect calorimetry was used to measure resting metabolic rate in healthy subjects. Skeletal muscle biopsies were extracted from vastus lateralis for isolation of myogenic satellite cells and protein analysis. Myotubes were transfected with an expression vector in order to achieve over expression of COX IV‐2 and with siRNA for knocking down COX IV‐1 (COX IV‐1−/2+). The control cells were transfected with a control vector and siRNA respectively. Respirometric measurements and hydrogen peroxide production were assessed simultaneously in an oxygraph (O2‐K, Oroboros) equipped with a spectrofluorometer by using the fluorophore Amplex®UltraRed.

Results

Here we show that COX IV‐2 protein is expressed in skeletal muscle harvested from healthy humans with a large inter‐individual difference in the COX IV‐2/COX IV‐1 ratio. A strong inverse correlation was found between the COX IV‐2/COX IV‐1 ratio and RMR measured in the same individuals. The same pattern was observed in the primary human skeletal myotubes overexpressing COX IV‐2 with knocked down COX IV‐1, showing a >60% lower basal cell respiration and a >50% reduction in LEAK respiration. The latter suggests either a lower proton conductance or a reduction in proton slip at COX. In addition, reduced cellular H2O2 production was observed both during LEAK and basal cell respiration.

Conclusion

These results suggest an important role of the mitochondrial subunit isoform COX IV‐2 in control of energy expenditure and mitochondrial reactive oxygen species homeostasis in humans.

Support or Funding Information

This study was supported by grants from the Swedish Research Council, Swedish Heart and Lung Foundation, Stockholm City Council (ALF), Swedish National Centre for Research in Sports and funds for the Karolinska Institutet.
Distribution pattern of dietary protein intake does not affect anabolic response, lean body mass, muscle strength or function over 8 weeks in older adults
Authors: Kim Il‐Young , Schutzler Scott , Azhar Gohar , Wolfe Robert , Ferrando Arny
Published: November 2023

Our recent acute study of dietary protein distribution indicated no difference in the anabolic response to differing patterns of dietary protein intake. However, it has been argued that acute metabolic studies may not predict functional changes over a longer period time. To examine the chronic effects of protein distribution pattern, older subjects (51– 69 yrs) consumed 1.1 g/kg/d in 2 distinct patterns. One group (n=7) consumed protein in a traditional, or uneven, manner (UNEVEN, 15/20/65%; breakfast, lunch, dinner), while the other group (n=7) consumed meals with an even distribution of protein (EVEN: 33/33/33%). Lean body mass (LBM), functional outcomes, and protein kinetics at whole‐body and muscle levels using stable isotope tracers were determined before and after 8 weeks of each respective protein intake pattern. There were no significant differences between the intake patterns in LBM, muscle strength, and other functional outcomes. Consistent with these findings, the 20‐h integrated whole body protein kinetics [net protein balance (NB), protein synthesis (PS), and breakdown (PB)] above basal states and muscle protein synthesis were not different between the intake patterns. We conclude that the distribution pattern of protein intake in mixed meals over an 8‐week period does not affect protein metabolism, lean body mass, muscle strength, or functional outcomes.

Support or Funding Information

The project was financially supported by a grant from National Dairy Council, Egg Nutrition Center, and National Cattlemen's Beef Association. This project was financially supported by Pepper Center Grant PG30‐AG‐028718 and Award Number UL1‐TR‐000039 and KL2‐TR‐000063 from the National Center for Advancing Translations Sciences (NCATS).
Nutritional Status Affects Expression of AMPK mRNA in a Tissue and Subunit‐specific Manner in Channel Catfish
Authors: Evans Paige , Vides Melissa , Teel Brianna , Kobayashi Yasuhiro
Published: November 2023

The objective of this study was to determine whether AMP Protein Kinase (AMPK) expression is associated with changes in food intake in channel catfish. AMPK is known as the master regulator of nutrient metabolism and has been associated with the development of obesity and other metabolic disorders. An obese‐like phenotype develops in channel catfish genetically selected toward an increased growth rate, suggesting that channel catfish could be a model organism to study the development of obesity and other metabolic disorders. However, the mechanisms associated with the development of the phenotype are unclear. After 28 days, brain, liver and muscle tissues were collected from juvenile catfish that were randomly assigned to one of three different treatments: fasted, fed and fasted‐refed. Expression of mRNA encoding AMPK beta, gamma, and alpha subunits were quantified using real‐time PCR. Expression of AMPK gamma and alpha 2 subunit mRNA in muscle, brain and liver were similar among all treatments (p>0.10). Expression of AMKP beta 1 and 2 subunit mRNA was also similar in muscle among the three treatments (p>0.10). In contrast, expression of AMPK beta 1 (p<0.01) and alpha 1 (p<0.05) subunit mRNA was higher in liver of fasted fish. The results of our study showed that, in channel catfish, changes in food intake influence the expression of AMPK mRNA in tissue‐and subunit‐specific manner. Given the importance of AMPK in the development of obesity, further studies are needed to identify the association between AMPK mRNA expression and the development of an obese‐like phenotype in channel catfish.

Support or Funding Information

This study is supported by the Kansas IDeA Network of Biomedical Research Excellence (grant number P20GM103418).
Long‐term Adaptations in Hepatic Substrate Utilization in 1 Year Old Rat Model of Bronchopulmonary Dysplasia
Authors: Levin Taylor S. , Tetri Laura , Haraldsdottir Kristin , Pegelow David F. , Braun Rudolf K. , Gross Kara N. , Eldridge Marlowe W.
Published: November 2023

Background

Approximately 12% of births in the United States are preterm, and this number is rising globally. Preterm birth is associated with increased long‐term risk for insulin resistance, metabolic syndrome, and cardiovascular disease. Infants born preterm often receive oxygen therapy, which, while life saving, can cause many changes in metabolism at the cellular level, especially in the liver. Hyperoxic exposure can inhibit processes such as glucose‐induced insulin secretion, which is linked to the inhibition of oxidative phosphorylation. We hypothesized that a two‐week postnatal hyperoxic exposure would result in a shift in metabolism from oxidative phosphorylation to a glycolytic pathway in the liver that persists into adulthood.

Methods

Harlan Sprague‐Dawley (SD) rat pups were exposed to either 21% oxygen (NORM, n=8; 3 males) or 85% oxygen (HYP, n=13; 6 males) for 14 days beginning within 12 hours of birth. After this 14‐day period, they were placed in room air and aged out to 1 year in standard normoxic conditions, at which point their livers were harvested and flash frozen in liquid nitrogen. Alterations in the expression of key metabolic regulators were analyzed by Western blotting. Key metabolic regulators include phosphofructokinase 1 (PFK‐1), lactate dehydrogenase A (LDHA), pyruvate dehydrogenase (PDH), and phosphorylated PDH (pPDH). The effects of sex and oxygen levels were compared using two‐way ANOVA with Tukey's for multiple comparisons.

Results

Expression of LDHA was increased in male HYP rats as compared to the male NORM group (P=0.02; increase of 36.9%). PDH was downregulated in the male HYP rats as compared to the male NORM rats (P<0.001; decrease of 34.8%). Finally, PFK‐1 is downregulated in the HYP females as compared to NORM females (P=0.001; decrease of 49.9%). There was no significant change in the pPDH/PDH ratio in either sex.

Conclusions

PDH converts pyruvate to acetyl‐CoA at the end of glycolysis. Acetyl‐CoA is then used in the TCA cycle to carry out respiration and energy formation. The downregulation of PDH seen in the HYP male group suggests that there is less cellular respiration as a result of postnatal HYP exposure. Together, the upregulation of LDHA and downregulation of PDH suggest that pyruvate is shunted away from the TCA cycle to form lactate. Alternatively, this could represent a downregulation of the TCA cycle and an increased utilization of lactate for gluconeogenesis. Overall, these findings suggest an increase in anaerobic metabolism and a decrease in aerobic metabolism within the liver following postnatal HYP exposure.

Support or Funding Information

Funding source: NHLBI, R01 HL115061‐03, Suppl (Eldridge) & NHLBI, R01 HL115061 (Eldridge)
Depletion of murine microbiota with broad‐spectrum antibiotics alters intestinal motility, secretion, and mucosal barrier function
Authors: Lendrum Jonathan , Seebach Bradley , Liu Sumei
Published: November 2023

The commensal microbiota in the gut coevolved with the mammalian host and is essential for many host physiological processes. Alterations in the composition of gut microbiota seem to be a pathogenic component of functional gastrointestinal disorders. The aim of this study is to investigate if a state of antibiotics‐induced intestinal dysbiosis is able to modify intestinal motility, secretion, and mucosal barrier function. C57BL/6 mice received a broad‐spectrum antibiotic cocktail (ampicillin 25mg/kg, vancomycin 10mg/kg, neomycin 25 mg/kg, and metronidazole 25 mg/kg) by oral gavage once a day for two weeks. Gastrointestinal transit was measured using a charcoal meal method. Ussing chamber techniques were used to assess colonic mucosal ion secretion and transepithelial electrical resistance (TER). Intestinal permeability to macromolecules was assessed by the flux of horseradish peroxidase (HRP, 44kD) from mucosa to serosa side of the colonic mucosa/submucosa preparations. Two weeks of antibiotics treatment delayed gastrointestinal transit. Colonic baseline ion secretion was increased in antibiotics‐treated mice. Antibiotics treatment also caused a dramatic increase in intestinal permeability, which was indicated by a significant reduction of TER and an increase in HRP flux from mucosa to serosa side of the colon. The results of this study suggest that depletion of intestinal microbiota with broad‐spectrum antiobiotics reduces gastrointestinal motility, enhances colonic ion secretion, and impairs mucosal barrier function and may contribute to functional gastrointestinal disorders.

Support or Funding Information

NIH R15 DK097460‐01A1 (SL) and UW‐L undergraduate research grant (JL)
Molecular changes leading to the abnormal migration of sacral neural crest cells in the Dominant megacolon mutant, a mouse model of Hirschsprung's disease
Authors: HUANG Taida , HOU Yonghui , CHAN Wood Yee
Published: November 2023

Although the majority of neurons and glia of the enteric nervous system (ENS) are derived from neural crest cells (NCCs) at the vagal (cervico‐thoracic) level, NCCs at the sacral level are also able to migrate to the hindgut of the developing embryo and contribute significantly to the ENS of the colon. Abnormal migration of NCCs may lead to a loss of enteric neurons and glial cells in humans, which is called Hirschsprung's disease (HSCR), and also in the Dominant megacolon mouse (Dom), which is an animal model of HSCR carrying a spontaneous mutation of Sox10. In this study, we aimed to follow the migration of sacral NCCs in the mutant embryos and determine the molecular events that lead to the abnormal migration of sacral NCCs. It was found with in situ cell labeling and whole embryo culture that migrating sacral NCCs tended to aggregate, resulting in their delayed migration toward the hindgut. Genome‐wide profiling of RNA expression with microarrays showed that the expression of the adhesion molecule cadherin 19 (Cdh19) was drastically down‐regulated in mutant sacral NCCs as compared to that in the wild‐type. Results of in situ hybridization with wild‐type embryos at E9.5 to E11.5 indicated that Cdh19 was localized to migrating sacral NCCs and their derivatives which also expressed Sox10. With luciferase assay and chromatin immunoprecipitation, the expression of Cdh19 was found to be directly regulated by Sox10, and the binding site of Sox10 on the promoter of Cdh19 was also identified. When the expression of Cdh19 was knockdown in the primary culture of wild‐type sacral NCCs, their migration ability was reduced, and conversely, upon re‐expression of Cdh19 in the mutant sacral NCCs, their migration ability was restored to a level close to that of wild‐type sacral NCCs. Further experiments with immunoprecipitation showed that the Cdh19 expression modulated NCCs migration via interacting with catenins which are important regulators of the dynamic cytoskeleton of migrating NCC. In summary, the present study showed that the Sox10 mutation in the Dom mutant resulted in the delayed migration of sacral NCCs and significant down‐regulation of the expression of Cdh19 which was a direct downstream target of Sox10. Alterations of Cdh19 expression changed the migration ability of sacral NCCs through interactions with catenins which in turn regulated the dynamic cytoskeleton of migrating NCCs.

Support or Funding Information

This work was supported by a RGC General ResearchFund (CUHK14102214) and a Direct Research Grant from the Faculty of Medicine, TheChinese University of Hong Kong (2014.1.074).
Lmx1b‐Mediated Limb Dorsalization: Identification of a Potential Regulatory Sequence for Keratocan, Lumican, and Decorin
Authors: Spady Rachael Nicole , Haro Endika , Pira Charmaine U , Feenstra Jennifer M , Oberg Kerby C
Published: November 2023

Lmx1b is a homeodomain transcription factor expressed within the dorsal limb mesoderm. Lmx1b knock‐out mice develop ventral‐ventral limbs, while ventral overexpression results in dorsal‐dorsal limbs. Our goal is to further characterize Lmx1b‐regulated limb dorsalization by identifying downstream target genes. Gene array studies show up‐regulation of the extracellular matrix genes Keratocan (Ker), Lumican (Lum), and Decorin (Dcn) in the presence of Lmx1b. These three genes are clustered within 134k bp of each other on chromosome 11 in mice. Lmx1b‐targeted chromatin immunoprecipitation followed by massively parallel sequencing (Lmx1b‐ChIP‐seq) identified a potential regulator sequence (PRS), 509 bp in length, located 929 kb downstream of Lum with 3 potential Lmx1b binding sites. We reasoned that Lmx1b binding to this PRS might regulate Ker, Dcn and/or Lum expression during limb dorsalization.

To determine activity, the PRS was isolated by PCR, incorporated into a construct with a basal promoter linked to GFP reporter, and then electroporated into the presumptive wing field of stage 14 chick embryos. We co‐transfected a β‐actin promoter‐driven RFP construct with the PRS reporter to confirm transfection efficiency. To determine the requirement for Lmx1b's binding within the PRS, we repeated the experiment following site directed mutagenesis of the predicted Lmx1b binding sites.

The PRS is active in the ventral mesoderm of the developing limbs and absent in dorsal limb mesoderm proximally. This suggested that Lmx1b might inhibit activity of this PRS. However, there was no apparent change in enhancer activity after site‐directed mutagenesis of the 3 Lmx1b binding sites. Thus, Lmx1b is not likely to play a direct role in restricting this enhancer from dorsal mesoderm. Further analyses will be required to determine the factors responsible for this unique pattern of enhancer activity.

Support or Funding Information

Loma Linda University; National Organization of Rare Diseases (NORD)
Receptors for Advanced Glycation End‐products (RAGE) inhibition protects from Intrauterine Growth Restriction (IUGR) during Second Hand Smoke (SHS) in Mice
Authors: Jordan Clinton J , Lewis Josh B , Bodine Jared S , Lewis Adam L , Dunaway Todd M , Egbert Kaleb M , Monson Troy D , Ogden Connor , Wright Tanner J , Mejia Camilo A , Reynolds Paul R , Arroyo Juan A
Published: November 2023

Intrauterine growth restriction (IUGR) is a disease affecting 10% of all pregnancies and is often characterized by hypoxia, asphyxia, and fetal demise. Primary and secondhand cigarette smoke (SHS) during pregnancy is known to induce IUGR. Receptors for Advanced Glycation End‐products (RAGE) are transmembrane receptors increased by SHS in the placenta and RAGE signaling is related to the induction of inflammation. We tested the hypothesis that inhibition of RAGE during SHS exposure protects from smoke‐induced decreases in fetal growth. C57/Bl6 mice (n=8) were exposed to SHS alone or SHS + semi‐synthetic glycosaminoglycan ethers (SAGEs; an inhibitor of RAGE) for 4 days from day 14 to day 17 of gestation (dGA). RAGE −/− mice were also exposed to SHS for 4 days from day 14 to day 17 dGA. At the time of necropsy (18 dGA) placental and fetal weights were recorded and tissues were immediately frozen for histological and protein analysis. To address the effects of the RAGE inhibition and cigarette smoke in trophoblast cell invasion, first trimester trophoblast cells were treated with cigarette smoke extract (CSE) alone or CSE in the presence of SAGEs for 24 hours. Mice treated with SHS alone demonstrated a 4.6‐fold reduction (p<0.001) in fetal weight and 1.4‐fold reduction in (p<0.002) in placental weight when compared with controls. When mice were co‐treated with SHS and SAGEs, a reduction of SHS‐induced fetal weights (1.7‐fold, p<0.001) and placental weights (1.3‐fold, p<0.03) were determined when compared to controls. When RAGE −/− mice were exposed to SHS, no differences in fetal weights and placental weights were determined. Invasion of trophoblast cells decreased 92% (p<0.002) when treated with CSE for 24 hours. This inhibition of invasion was completely reversed when SAGEs were added to the CSE media added to trophoblasts. We concluded that inhibition of RAGE protects against fetal and placental weight loss during SHS‐induced IUGR. This conclusion was also supported by the discovery that hindered trophoblast invasion was reversed by SAGE treatment. Our results further suggested that there is a direct correlation between RAGE activation and the development of IUGR during SHS. These studies provide insight into tobacco‐mediated IUGR progression and clarify possible avenues for alleviating placental complications during SHS exposure.
The Effects of Staphylococcus aureus Biofilm Conditioned Medium on Fibroblast Tension Generation and Migration
Authors: Kshetri Pratiksha , Brennan Robert E , Vaughan Melville B
Published: November 2023

Chronic wounds are often characterized by persistent inflammation. These wounds provide a favorable environment for bacteria to establish an infection, which may lead to the formation of a biofilm that can delay the healing process. Although the occurrence of biofilms in chronic wounds is known, the role of these biofilms in chronic wound pathogenesis is still unclear. Chronic wounds may be affected directly by the biofilm or through interaction with secreted products of the biofilm bacteria. Our goal was to investigate the Staphylococcus aureus biofilm extract effects in collagen lattice tension generation on fibroblasts. The control and TGF‐beta treated lattices were cultured in presence or absence of S. aureus biofilm conditioned medium. The mature control lattices showed the greatest contraction followed by biofilm with less contraction. The reduction of contraction was correlated with less number of proliferated and differentiated myofibroblasts in biofilm‐conditioned treated lattices. Tension generation is one of the important factors in wound closing by myofibroblasts; cell migration is another important aspect of wound healing. We also hypothesized that the biofilm conditioned medium of S. aureus would affect the migration of fibroblasts. The quantitative analysis of two‐dimensional cell migration was conducted using silicone elastomer mask migration assay. Cells were cultured in the presence or absence of biofilm conditioned medium and the migratory response was measured with the image of cell free area invaded by fibroblasts at T0, T24, T48 and T72 hrs. The percentage of area invaded by fibroblasts in control at T48 was found to be 62.04 whereas the percentage of area invaded for biofilm‐treated cells was 58.05. In conclusion, fibroblast tension generation and migration behavior was inhibited by the biofilm conditioned medium. The future goal is to investigate the mechanism behind the inhibition properties of biofilm conditioned media on fibroblasts.

Support or Funding Information

3M Corporation and UCO Office of Research and Grants
Investigation of Anterior Cruciate Ligament Fibroblast Biomarkers for Cell Characterization by Immunohistochemistry
Authors: Lerma Tanya , Guttmann Geoffrey D.
Published: November 2023

Approximately 200,000 anterior cruciate ligament (ACL) injuries occur in the United States per year. This ligament is also the most commonly injured ligament of the knee joint. Surgical reconstruction using autografts from the patient is the main method of treatment to date; however, cadaver tendon allografts may also be used. A less invasive and more promising method for repair is ligament engineering. Growth factors, a proper matrix, and suitable cells, are a few of the necessary components of a ligament construct.

A common protocol to engineer a ligament is to use ACL fibroblasts and to seed them into a matrix or scaffold that allows the creation of a similar tissue. However, to date no successful construct has been engineered. In order for an engineered construct to be successful and resemble a human ACL, an understanding of the cellular components of the ACL fibroblasts is necessary. The existence and role of the cellular components in the human ACL allows for the ligament to maintain its mechanical. Therefore, it is necessary to investigate every aspect of this tissue, including using reliable biomarkers to properly characterize human ACL fibroblasts. In this study, we aim to establish a combination of biomarkers specific to these fibroblasts using the method of immunohistochemistry.

The central hypothesis of this study is that the biomarkers of interest, vimentin, fibronectin, alpha‐smooth muscle actin(α‐SMA), Fibroblast Growth Factor Receptor 1 (FGFR1), and collagen III will be a suitable and reliable combination of markers to characterize anterior cruciate ligament (ACL) fibroblasts because 100% of the cells are expected to stain positive for these biomarkers by the method of immunohistochemistry.

Our results indicate that none of the biomarkers were detected in cells at a rate of 100%. Fibronectin and collagen III abundance in the ECM made it difficult to determine specific production by an isolated fibroblast. However, both biomarkers were detected in abundance by fluorescence intensity. FGFR1 and vimentin stained positive in over 50% of cells, but less than 95%. Alpha‐SMA was more variable due to two of the ligaments having 0% of cells stain positive for this biomarker. However, samples that did show expression of alpha‐SMA stained over 46% of cells.
Olive Oil Administration Improves Cutaneous Wound Healing of Pressure Ulcers in Mice
Authors: Alto Costa Andréa Monte , Donnato‐Trancoso Aline , Romana‐Souza Bruna
Published: November 2023

Chronic lesions, as pressure ulcers, present high levels of reactive oxygen species (ROS) and exacerbate inflammatory response, which impair cutaneous wound healing. Olive oil has anti‐inflammatory and antioxidant proprieties and its administration in chronically stressed mice may improve the cutaneous wound healing. The aim of this study was to investigate the effects of olive oil administration on cutaneous wound healing of pressure ulcers in mice. For this, male mice were daily treated with olive oil (1.5 g/kg) until euthanasia. One day after the beginning of treatment, two cycles of ischemia–reperfusion by external application of two magnetic plates were performed in skin to induce pressure ulcer formation. All experimental animal work was approved by the Ethical Committee for Animal Use of State University of Rio de Janeiro (CEUA/044/2014). The olive oil administration increased the wound contraction 7 days after ulceration when compared to control (water‐treated) group. The re‐epithelialization was increased by olive oil administration when compared to control group 7 and 14 days after ulceration. The ROS levels on wound area was increased 3 days after ulceration, but decreased 7 days, in olive oil‐treated group when compared to control group. The olive oil administration reduced the levels of lipid hydroperoxides when compared to control group. In conclusion, the administration of olive oil accelerates ROS synthesis and wound closure of pressure ulcer in mice.

Support or Funding Information

Financial support: Carlos Chagas Filho Foundation for Research Support in the State of Rio de Janeiro (FAPERJ).