bluuebunny/crossref_metadata_2025 huggingface dataset:
C‐kit cardiac precursor cells exhibit a unique Nox isoform profile which may influence their survival, proliferation, and differentiation in the infarcted heart
Authors: Spealman Alyson Korry , Poor Daniel A. , Sharma Ram V. , Kotlikoff Michael I. , Davisson Robin L.
Published: June 2021

Compelling evidence now confirms the presence of small but distinct populations of stem cells residing in niche areas of the adult heart. Evidence from our laboratory demonstrates that c‐kit‐positive cells isolated from the hearts of c‐kitBAC‐EGFP neonatal mice can be expanded and differentiated in vitro into all three cell lineages of the heart. Since NADPH oxidases (Nox) play critical roles in cardiomyogenesis and stem cell survival, and also have distinct roles in cell signaling and death, we hypothesized that c‐kit cells exhibit a unique Nox profile compared to control neonatal cardiomyocytes. Utilizing real‐time RT‐PCR and expressing the data relative to β‐actin, our results indicate that Nox1 is markedly upregulated in c‐kit cells compared to control cardiomyocytes (13.40±4.34 fold, n=3, p<0.05). Nox4 is also upregulated, but to a lesser extent than Nox1 (1.73±0.45 fold, n=3). On the other hand, Nox2 is significantly downregulated compared to control cardiomyocytes (.37±.16 fold, n=3, p<0.05). These data reveal a highly unique Nox profile in c‐kit‐positive progenitors, and may provide genetic targets for improving cell survival, proliferation, and/or differentiation following cardiac damage. Supported by HL063887, HL084624, AHA 0540114N.
Insulin Therapy Effects on Bone Health in Rats Following Severe Burn and Disuse
Authors: Baer Lisa A , Pidcoke Heather F , Wu Xiaowu , Silliman David T , Walters Thomas , Tou Janet , Wolf Steven E , Wade Charles E
Published: June 2021

Burn with hindlimb unloading (BH) results in substantial and sustained bone loss in rodents. We observed decreases in bone mineral density (BMD), content (BMC), and strength using this model. Insulin used for glucose control in burns alters burn induced hypermetabolic responses. The present study was conducted to determine if daily insulin treatment could offset effects of BH on body mass, bone mass and morphology. Male rats were randomized into two groups: BH‐Vehicle (BHV) or Insulin‐Treated (BHI). Rats received a 40% total body surface area scald burn and following recovery immediately hindlimb unloaded. Daily subcutaneous injections were either long‐acting insulin or 0.9% saline. Fourteen days after injury body mass, visceral fat, femur weight and length were measured, and BMD and BMC were determined by DEXA. Body mass and fat were significantly heavier in BHI. No differences were observed in femur weight, length or BMD, however BHI showed a significant decrease in BMC. Even though insulin sustained body mass and fat, there were minimal effects on bone. Insulin therapy may not be effective in maintaining bone health in burn patients. US Army MRMC & Juvenile Diabetes Foundation
Endothelial modulation of swine coronary artery tone through coupling of basal IP3R‐dependent Ca2+ signals to endothelial SK and IK channels
Authors: Francis Michael , Solodushko Viktoriya , Taylor Mark S
Published: June 2021

Basal Ca2+ events originate from IP3 receptors (IP3R) at distinct sites in endothelial cells leading to stimulation of membrane Ca2+‐activated K+ channels (KCa). Persistent activation of small (SK) and intermediate (IK) conductance KCa channels may provide the foundation of endothelium derived hyperpolarization (EDH) in many vascular beds. Here we investigate the role of IP3‐mediated Ca2+ signaling through SK or IK channels in endothelial modulation of sustained coronary artery tone. In arteries pre‐contracted with U46619, the SK channel blocker apamin and the IK channel blocker charybdotoxin increased tone by 28.2% and 40.0%, respectively. These effects were endothelium dependent and were completely blocked by pretreatment with IP3R inhibitors xestospongin C or 2APB but not by ryanodine. Confocal imaging revealed dynamic endothelial Ca2+ events emanating from distinct spots along the basolateral membrane. Immunostaining in the same vessels showed endothelial expression of all three IP3R isoforms with discrete distribution profiles. Our findings indicate basal endothelial IP3R Ca2+ signals continuously modify coronary artery tone through constitutive SK and IK dependent hyperpolarization and relaxation. A working deterministic model was generated relating the density and distribution of specific endothelial IP3Rs to the origination and dynamics of Ca2+ events in coronary artery endothelium.
Weight loss attenuates oxidative stress related‐coronary vasoconstriction in obese adolescents
Authors: Gao Zhaohui , Momen Afsana , Novick Marsha , Williams Ronald J. , Cyran Stephen E. , Blaha Cheryl , Mast Jessica , Spilk Samson , Leuenberger Urs A. , Sinoway Lawrence I.
Published: June 2021

Obesity is a risk factor for cardiovascular disease. Obesity is also a disease of oxidative stress (OS) in which cardiac output and myocardial oxygen consumption is increased. We have shown that hyperoxia (breathing 100% O2; H) can evoke coronary vasoconstriction by the oxidative quenching of NO. To examine if weight loss (WL) would alter the H related‐coronary constriction seen in obese adolescents, we measured coronary blood velocity (CBV) during H using Doppler Echocardiography before and after a 4‐week diet‐and‐exercise regimen in 6 obese male adolescents (age 15 ~ 18 yrs, BMI, 36.5±5.7 kg/m2). Three age and BMI‐matched controls were also studied. Coronary vascular resistance (CVR) was calculated as diastolic blood pressure (BP)/CBV. WL (10.1±3.1 kg) was associated with a reduction in BP, HR and rate‐pressure product (RPP) (all P<0.05). Before weight loss H increased CVR by 37±6%, whereas after WL CVR increased by only 19±6% (P<0.05) with H. CVR responses remained constant in the 3 time control subjects. Thus weight loss: 1) reduces HR, BP, and RPP; and 2) attenuates the OS related‐coronary constriction seen in obese adolescents. We speculate that: 1) the higher RPP before WL led to a higher NO production and impressive H induced constriction; 2) after WL, NO levels were reduced (less coronary flow due to lower RPP) and the effects of H were attenuated.

Supported by R01 HL070222 (LS) and Tobacco Settlement Funds (LS).
Can leptin induce fever?
Authors: Steiner Alexandre A , Krall Catherine M , Liu Elaine
Published: June 2021

It is unclear whether physiological acute rises in circulating leptin (an adipocyte‐derived hormone) can cause fever. We studied the effects of recombinant leptin on the body temperature (Tb) and plasma leptin of rats that were food‐deprived (lower baseline leptin) or free‐feeding (higher baseline leptin). The biological activity of leptin was confirmed by its ability to suppress food intake. In food‐deprived (24 h) rats kept in a cool environment (22 °C), iv infusion of leptin (250 μg/kg) elevated Tb from hypothermic to normothermic values, but it did not raise Tb to febrile values. Such an effect coincided (120 min post‐infusion) with a rise in plasma leptin to a level (7.8 ± 0.6 ng/mL) often found when the endogenous production of leptin is activated. Increasing the leptin dose to 1,000 μg/kg did not further elevate Tb in food‐deprived rats. In free‐feeding rats kept in a cool (22 °C) or warm (28 °C) environment, leptin infusion did not alter Tb, even when a dose of 3,500 μg/kg raised plasma leptin to a level (75.2 ± 12.6 ng/mL at 120 min) that is substantially above the endogenous level of leptin in leptin‐responsive rats. We conclude that an acute rise in plasma leptin does not induce fever in rats.

Support

Scholarship of discovery grant, Albany College of Pharmacy & Health Sciences
Preoptic Nitric Oxide Modulates Endotoxic Fever by Controlling Local Norepinephrine Activity
Authors: Feleder Carlos , Perlik Vit , Blatteis Clark M
Published: June 2021

Lipopolysaccharide (LPS) injected iv stimulates norepinephrine (NE) release in the preoptic‐anterior hypothalamus (POA). Since NE upregulates neuronal nitric oxide (NO) synthases and NO induces cyclooxygenase (COX)‐2‐dependent prostaglandin (PG)E2, we investigated whether NO mediates the production of COX‐2‐catalyzed PGE2 induced by the NE activation of POA α2‐adrenoceptors (AR). We injected LPS (2 μg/kg iv) into guinea pigs prepared with intraPOA microdialysis probes and, continuously microdialyzed SIN‐1 (NO donor, 20 μg/ μl) or carboxy‐PTIO (NO scavenger, 20 μg/ μl). POA interstitial fluid NE, PGE2 and core temperature (Tc) were measured. We also microdialyzed clonidine (α2‐AR agonist, 2 μg/μl) with and without SIN‐1 or carboxy‐PTIO, or (+)‐catechin (an antioxidant, 3μg/μl) into the POA of other animals, and likewise determined POA PGE2 and Tc. SIN‐1 and carboxy‐PTIO depressed and augmented, respectively, the rises in POA NE, PGE2 and Tc produced by iv LPS. Similarly, they prevented and increased, respectively, the delayed elevations of POA PGE2 and Tc induced by clonidine. (+)‐Catechin prevented LPS‐induced elevation of PGE2, but not that of Tc. We conclude that NO is an inhibitory modulator of LPS‐induced release of POA NE, acting as a central antipyretic. These data also implicate free radicals in the formation of preoptic PGE2 and question its obligatory role in fever production.
5′‐AMP and its Role in Fasting‐Induced Torpor and Hypothermia in Ecto‐nucleotidase Knockout (CD73 ‐/‐) Mice
Authors: Eisner Christoph Benedikt , Qin Yan , Thiel Manfred , Briggs Josephine , Schnermann Jurgen
Published: June 2021

Fasting of mice induces a state called torpor that is accompanied by a reduction of core body temperature (CBT) at ambient temperatures (Ta) lower than thermoneutrality. It was recently shown that fasting raises 5′‐AMP (AMP) levels in plasma and an injection of AMP induces reversible profound hypothermia in mice. To assess whether AMP itself or its breakdown product adenosine is responsible for AMP‐induced hypothermia we compared the effect of AMP in wild type (WT) and CD73‐/‐ mice subjected to 48 h of fasting at a Ta of 22 °C while recording CBT by radiotelemetry. Both genotypes exhibited two hypothermic bouts with nadirs at 12 (WT: 31.9 ± 0.8 °C, CD 73 ‐/‐: 29.7 ± 1.4 °C; n=7) and 36 h (WT: 31.1 ± 0.6 °C, CD 73 ‐/‐: 28.2 ± 1.5 °C; n=7) after the start of fasting. The trend of a more profound torpor bout in CD73 ‐/‐ was also seen when data were expressed as area under curve (WT 8260 ± 1124 °C·min, CD 73 ‐/‐ 12683 ± 3425 °C·min; n=7). Similarly, injection of AMP (550 mg/kg BW) produced a significantly lower nadir of CBT in CD73 ‐/‐ than WT mice (26.9 ± 0.5 °C vs. 28.7 ± 0.4 °C; p < 0.05). Since plasma adenosine was not significantly altered by AMP in CD73‐/‐ mice (311 ± 21 pmol/ml vs. 295 ± 67 pmol/ml) whereas it increased to 1345 ± 287 pmol/ml in WT, it appears unlikely that hypothermia is caused by adenosine. In conclusion, hypothermia following fasting or AMP administration is caused likely by AMP itself and not by its extracellular conversion to adenosine.
New method of calculating alveolar CO2 transport
Authors: Caucha Luis Jhony , Cruz Julio Cesar , Rueda Luis Antonio
Published: June 2021

Cruz proposed that the expirogram of CO2 does not represent the process of gas exchange (FASEB J. 21:609.4, 2007). It represents a simple alveolar gas dilution by the tidal volume that does not reach the residual volume (RV). Caucha et al. showed that phase II is formed by the distribution of regional dead spaces (FASEB J. 22:763.6, 2008). However, this latter model could not predict which anatomic Weibel's model generations were involved. Thus, here, we modify Caucha's model by separating the convection‐diffusion process (method 1) in three different steps (method 2): 1. Convection. 2. Convection‐diffusion and 3. Diffusion. As a result, new alveolar CO2 concentrations at the apex and base regions of the lung were found (see figure, interrupted and continuous lines for method 1 and 2, respectively). This picture was obtained after searching the generations involved: the first 8 generations (step 1), generations 9th‐19th (step 2) and the last 5 generations (step 3), which produced the simulated expirogram similar to the experimental curve. In conclusion, the analysis presented follows our theory of alveolar gas mixing; during inspiration, the alveolar CO2 is diluted by the tidal volume (figure), and during expiration, the uneven emptying of parallel regions of the lung generates the expirogram.

Supported in part by ONG ‐ Centro de Enseñanza Investigación y Servicios.
HIF‐2α down‐regulation by intermittent hypoxia in rats induces oxidative stress resulting in autonomic dysfunction
Authors: Nanduri Jayasri , Wang Ning , Yuan Guoxiang , Khan Shakil , Souvannakitti Dangjai , Peng Ying‐Jie , Kumar Ganesh K , Garcia Joseph A
Published: June 2021

Humans with sleep‐disordered breathing (obstructive sleep apnea, OSA) experience chronic intermittent hypoxia (IH) and exhibit increased sympathetic activity and elevated blood pressures. Previous studies reported that increased oxidative stress resulting from IH contributes to autonomic dysfunction in rodent models of IH. Our observation in PC12 cells, that IH down‐regulates HIF‐2α via calpain proteases resulting in oxidative stress prompted us to examine the role of HIF‐2α down‐regulation in IH‐evoked autonomic changes. HIF‐2α was down‐regulated in carotid bodies and adrenal medullae from IH exposed rats. IH increased calpain activity and decreased SOD2 activity in the adrenal medulla of IH rats resulting in oxidative stress. Systemic treatment of IH‐exposed rats with calpain inhibitor ALLM rescued HIF‐2α degradation and restored SOD2 activity and prevented oxidative stress. More importantly, arterial blood pressures and plasma norepinephrine levels, which were significantly elevated in IH exposed rats, were restored to control values following ALLM treatment. The present observations on the effects of HIF‐2α degradation by IH provide new insights into the molecular mechanism(s) underlying IH‐evoked oxidative stress and the resulting autonomic morbidities associated with recurrent apneas. Supported by NIH‐HL‐90554 and HL‐76537.
Overexpression of adipogenin enhances the fat accumulation of C2C12 myoblasts
Authors: Song Sang‐Houn , Roh Sang‐Gun , Katoh Kazuo
Published: June 2021

Adipogenin is known that its expression is higher in adipose tissue and increases during the adipocyte differentiation. However, the roles and mechanisms of adipogenin on fat accumulation and adipocyte differentiation remain largely unknown. In this study, we investigated whether constitutive overexpression of adipogenin enhances adipogenic differentiation and fat accumulation in C2C12 cells. Several clones expressing high levels of adipogenin were isolated and characterized. Cloned cells were proliferated to confluent, and differentiated with adipogenic differentiation medium. After induction of differentiation, cells were stained with Oil Red O and mRNA expression was measured at day 5. Fat accumulation was observed in adipogenin‐transfected C2C12 cells at 2 day after induction of differentiation and the lipid drops were larger at day 5. The expression of adipogenic maker genes, PPAR‐γ2 and leptin, was detected in cells transfected with adipogenin. These results demonstrate that overexpression of adipogenin in C2C12 enhances fat accumulation in vitro.
PPARγ‐Dependent Modulation of Gene Expression in SHR: Role of GRK‐2 Signaling
Authors: Yousefipour Zivar , Uwadia Ndidi , Oyekan Adebayo , Newaz Mohammad
Published: June 2021

Peroxisome proliferator‐activated receptor ƒ× (PPARγ), a nuclear transcription factor, regulates blood pressure (BP) via distinct signaling mechanisms that involve G‐protein coupled receptors (GPCRs). GPCR activity is tightly regulated by GPCR kinases (GRKs) and increased GRK‐2 protein and mRNA was observed in hypertension (HTN). PPARγ ligands reduce BP in HTN but their connection in GPCR signaling is unknown. We propose that PPARγ ‐dependent BP reduction involves GRK‐2 and/or signaling molecule Raf ‐1. Male SHRs and WKY (control) rats were treated with GW1929 (G) or clofibrate (C), a PPARγ and α ligand, respectively, for 2 months. Heart homogenate were used to assess PPARγ, GRK‐2 and Raf‐1 gene and protein expression. PPARγ protein but not mRNA was reduced in SHR and both G and C enhanced PPARγ protein and mRNA in SHR. GRK‐2 protein and mRNA were higher in SHR and this increase was blunted by G and C. G also increased PPARγ mRNA but reduced GRK‐2 protein in WKY rats whereas C was without effect. Raf‐1 protein and mRNA was unchanged in SHR although G enhanced the expression in both study groups. C increased Raf‐1 mRNA but not the protein in WKY rats without effecting SHR. We suggest that PPARγ activation regulates GRK‐2 gene in physiological and pathological situations by a mechanism that involves Raf‐1 gene in HTN. These data also suggest that PPARα activation selectively influenced GPCR signaling in HTN.
Bioaccumulation of carotenoids and sulforaphane conjugates from tomato and broccoli powders with differing bioactive profiles
Authors: Liu Ann G , Volker Sonja E , Jeffery Elizabeth H , Erdman John W
Published: June 2021

High consumption of tomatoes and cruciferous vegetables has been linked to decreased risk of prostate cancer. Previous studies have evaluated the cancer preventive potential of individual bioactives found in tomatoes and broccoli, but few have examined them within the context of a whole food. We determined bioavailability of carotenoids and sulforaphane from 3 tomato and 4 broccoli diets with differing bioactive compound profiles to assess their potential for future cancer trials. Male Copenhagen rats were pair fed diets containing 10% tomato powder, high lycopene tomato powder, high carotenoid tomato powder, broccoli floret powder, broccoli sprout powder, high indole‐3‐carbinol broccoli powder, or high selenium broccoli powder for 7 days. The high lycopene diet resulted in greater hepatic lycopene accumulation and lower levels of phytoene and phytofluene compared to standard tomato powder. In groups fed broccoli diets, sulforaphane N‐acetylcysteine conjugates were determined in urine. The broccoli sprout diet resulted in the highest level of urinary sulforaphane conjugates and a significantly greater recovery of 39.4%, compared to 5.1% from the broccoli floret diet. These results demonstrate differential bioavailability of carotenoids and sulforaphane from tomato and broccoli diets with varying bioactive profiles. Funded by the Illinois Council on Food and Agricultural Research CF071‐006‐4‐SEN.
Influence of chocolate formulation factors on in vitro bioaccessibility and bioavailability of catechins in humans
Authors: Neilson Andrew Peter , George Judy , Mattes Richard D. , Janle Elsa M. , Matusheski Nathan V. , Ferruzzi Mario G
Published: June 2021

Conflicting data exist regarding the effect of carbohydrate and milk on bioavailability of catechins from chocolate. To test the effects of these ingredients, solid [reference dark (SD), high sucrose (SHS), high milk solids (SMS)] and beverage [high sucrose (LHS), artificial sweetener (LAS)] chocolate products providing physiologically relevant levels of epicatechin (EC, 30 mg/serving) were formulated. Subjects (n=6) consumed one serving of chocolate, and serum pharmacokinetics were monitored for 6 h. Subjects consumed all 5 products in a crossover design. Plasma Cmax and AUC0‐6h of EC from SMS samples were not significantly different from the reference SD chocolate, but were significantly lower than SHS as well as both beverages. Plasma Cmax of EC from the LHS beverage was also significantly higher than the reference SD chocolate. Recovery of EC through in vitro digestion (bioaccessibility) did not differ between formulations. Furthermore, EC accumulation by Caco‐2 intestinal cells exposed to in vitro digesta from each product for 1 h were not significantly different, but retention of EC over 1 h was significantly greater in cells exposed to digesta from SMS product than other solid products. These data suggest that gastric emptying and intestinal cell retention are the main mechanisms by which formulation factors may alter the pharmacokinetic properties of catechins from chocolate. Supported by Kraft Foods.

Grant Funding Source

Kraft Foods Global
Unraveling the complexity of Echinacea fractions to identify alkylamides and ketones important for anti‐inflammatory bioactivity
Authors: LaLone Carlie A. , Rizshsky Ludmila , Solco Avery , Nikolau Basil , Murphy Patricia , Birt Diane F.
Published: June 2021

Third round fractions produced via bioactivity guided semi‐preparative reversed phase HPLC fractionation of Echinacea angustifolia were analyzed by GC‐MS to identify constituents important for the anti‐inflammatory properties of the botanical. Two fractions were of interest due to their ability to inhibit lipopolysaccharide induced PGE2 production in RAW264.7 mouse macrophage cells and their GC‐MS chromatograms having peaks for known and synthetically available compounds. When identification and quantification from GC‐MS revealed that major constituents present in one fraction (1 µg/ml) were alkylamide 10 (0.15 µM) and ketone 23 (0.24 µM), and that the other fraction (1 µg/ml) consisted of alkylamide 11 (3.6 µM), these constituents were screened at the concentrations present in the fraction using the PGE2 assay. The combination of alkylamide 10 and ketone 23 did not explain the bioactivity of its respective fraction, but alkylamide 11 was partially explanatory of the bioactivity, accounting for 30% reduction of PGE2 production compared to 81% reduction of PGE2 identified with the fraction. Chemically synthesized ketones 21 and 23 were also shown to individually inhibit PGE2 and nitric oxide production at a concentration as low as 1µM, providing a basis to study these constituents further for anti‐inflammatory potential. Funding: 9 P50 AT004155‐06 from ODS/NCCAM, NIH.

Grant Funding Source

9 P50 AT004155‐06 from ODS/NCCAM, NIH
Transcriptional regulation of the Menkes Copper Atpase (Atp7a) gene by iron.
Authors: Collins James F. , Lu Yan , Xie Liwei , Ranganathan P. N.
Published: June 2021

Copper and iron homeostasis‐related genes, Atp7a, metallothionein 1a (Mt1a) and divalent metal transporter 1 (Dmt1) are strongly upregulated in the intestine of iron‐deficient rats. A strong downregulation of hepatic hepcidin expression (>1500‐fold) was also noted. Atp7a and Dmt1 are induced throughout the length of the gut. We have modeled this observation in vitro in IEC‐6 cells, where both genes are induced by iron chelation. Utilizing Atp7a‐specific antibodies, we demonstrate that protein expression is also increased by iron deprivation, and that the protein seems to redistribute to different cellular compartments (i.e. from the trans‐golgi network to vesicular structures and the plasma membrane). Interestingly, actinomycin D inhibited the induction of Atp7a mRNA expression, indicating transcriptional regulation. We thus sought to determine the molecular mechanism of Atp7a gene induction by iron. We first mapped the 5' end of the transcript and found alternative splicing; all splice variants contained exon 1, but their 5' termini varied within a span of 50 bps. Promoter fragments were amplified by PCR off of a BAC genomic clone and cloned into a luciferase‐containing reporter gene vector. Preliminary studies demonstrated that the promoter responded to iron chelation, demonstrating that we have developed an appropriate model with which to determine the precise mechanism by which the Atp7a gene is regulated by iron deprivation.
BXD strains of mice exhibit diurnal fluctuations in brain and liver iron and iron management proteins
Authors: Unger Erica L , Hao Lei , Earley Christopher J , Beard John L
Published: June 2021

Restless Legs Syndrome patients frequently have a deficit in brain iron and show a significant diurnal variation in motor symptoms. Regulation of iron levels in the brain and flux of iron across the blood‐brain‐barrier are poorly understood, so the current study investigates diurnal fluctuations in plasma, liver and brain iron and in iron‐related proteins, including hepcidin and ferroportin. Plasma, spleen, liver, and ventral midbrain iron levels vary significantly between the light and dark phases of the diurnal cycle in several strains of BXD mice. The amount of variation is strain dependent (<10%‐to‐<30%) and corresponds to variations in iron management proteins (Tf, TfR, Ft). Hepatic and brain hepcidin mRNA levels also vary across the diurnal cycle and between BXD strains, suggesting a role for hepcidin regulation of iron flux at the BBB epithelial cell. Ferroportin, Ft, DMT1, and TfR are all located in these epithelial cells, and we believe one explanation for the large iron flux is an IL6 mediation of ferroportin gene expression. IL6 expression in brain is directly influenced by clock genes, putting in place a potential explanation for the novel observations of large diurnal variations in brain and liver iron. This work is supported by NIH grant P01‐AG021190.
Race/ethnicity differences in the relationship between obesity and gait speed among older Americans
Authors: Xu Beibei , Houston Denise , Gropper Sareen , Zizza Claire
Published: June 2021

Projections indicate that the population of older Americans will become more racially diverse. Health disparities cannot be addressed until differences in the associations between risk factors and outcomes are identified. Using data from the 1999‐2002 National Health and Nutrition Examination Survey, we examined the relationship between obesity, measured by BMI and waist circumference, and gait speed in 2,364 older adults (=60 y) and whether this relationship varies by race/ethnicity, gender, or age. Multiple linear regression models controlling for age, gender, race/ethnicity, education, physical activity, smoking status and standing height were used to estimate the association between obesity and gait speed. The relationship between BMI and gait speed differed by race/ethnicity. Among non‐Hispanic whites, overweight, obesity and extreme obesity all had significantly slower gait speeds (P = 0.006; P < 0.001; P < 0.001) when compared to normal weight. Among non‐Hispanic blacks, only those with extreme obesity had a significantly slower gait speed (P = 0.01) when compared to normal weight. No significant associations were observed among Hispanics. We observed similar results in the stratified waist circumference model. Our results may facilitate public health interventions by identifying those groups most vulnerable to the effects of obesity on physical functioning. Funded by Alabama Agricultural Experiment Station (ALA013‐020).
Effect of protein intake and weight loss on bone in overweight‐obese postmenopausal women
Authors: Sukumar Deeptha , Zurfluh Robert , Kwon Jane , Schneider Stephen , Stahl Theodore , Shapses Sue
Published: June 2021

High protein intake is a popular method to achieve weight (wt) reduction; however its effect on bone remains controversial. Understanding whether dietary protein intake influences bone mass during caloric restriction was the goal in this study while controlling for dietary calcium. We examined 40 postmenopausal women (58 ¡À 5 years; BMI of 32.4 ¡À 5.7 kg/m2), who were assigned to caloric restriction and 1.2 g Ca/day with either a normal (NP, 18%, n =20) or higher (HP, 30%, n =20) protein intake and a low fat intake (28%). Bone measurements using dual energy x‐ray absorptiometry were performed at baseline, and 6 and 12 months after wt loss. Here we report the data after 6 months of wt loss (‐7.3 ¡À 4%) that did not differ significantly between groups. Total body bone mineral density and lumbar spine bone mineral content decreased (p<0.05) more in the NP (‐2.0% and ‐1.6% respectively) compared to HP group (‐0.1 % and 1.2% respectively). In a subset of women (n=22), bone turnover markers were measured and showed no changes, but there was a trend (p¡Ü0.08) for a greater increase in serum 25‐hydroxyvitamin D and parathyroid hormone in the NP than HP group, that requires confirmation in the entire group. These data suggest that higher dietary protein attenuates bone loss during wt reduction in postmenopausal women. Further analysis of the mechanisms mediating these changes is being addressed.

SUPPORT: NIḦCAG12161, Busch Biomedical Grant

Grant Funding Source

NIA‐NIH
Long‐term caloric restriction increases apoptosis and decreases cell stress response, despite an elevation in antioxidant enzyme capacity in the skeletal muscle of the Cu/Zn‐SOD mutant G93A mouse, an animal model of ALS
Authors: Patel Barkha , Tarnopolsky Mark A , Hamadeh Mazen J
Published: June 2021

Despite its life‐extending properties, long‐term caloric restriction (CR) hastens clinical onset and shortens life span in the G93A mouse, an animal model of ALS (Hamadeh et al, 2005; Hamadeh and Tarnopolsky, 2005). We previously reported that CR increased lipid peroxidation (MDA) in this model (Patel et al, 2007). We hypothesized that CR would increase apoptosis and decrease cell stress response, with an incomplete compensatory upregulation of antioxidant enzymes. We investigated the effect of long‐term CR on the protein content of antioxidant enzymes (GPx1, GR), and markers of cell stress response (Hsp70) and apoptosis (Bax, caspase 9, cleaved caspase 9) in 99 d quadriceps of G93A mice. Starting at age 40 d, 27 mice were divided into ad libitum (AL, 14; 7 males) or CR (13; 7 males; 60% of AL). GPx1 increased in CR vs. AL mice (37%, P = 0.019) and in CR vs. AL females (56%, P = 0.012), with no change in GR. GPx1/GR was higher in CR vs. AL females only (88%, P = 0.013). Hsp70 was lower in CR vs. AL mice (62%, P = 0.002) and in males vs. females (37%, P = 0.030). Bax was higher in CR vs. AL mice (41%, P = 0.027) and in CR vs. AL females (52%, P = 0.048). Bax/Bcl‐2 was elevated in CR vs. AL mice (68%, P = 0.040) and in CR vs. AL females (2.3‐fold, P = 0.029). There were no differences in caspases. We conclude that CR increases apoptosis in the G93A mouse due to impairment in cell stress response and an incomplete compensatory upregulation of antioxidant enzymes.

Grant Funding Source

Hamilton Health Sciences Foundation, NSERC, Faculty of Health‐York University
Coffee increases antioxidant enzyme capacity in the brain of male G93A mice, an animal model of amyotrophic lateral sclerosis (ALS)
Authors: Seevaratnam Rajini , Raha Sandeep , Tarnopolsky Mark A , Hamadeh Mazen J
Published: June 2021

Oxidative stress is implicated in several neurodegenerative diseases including ALS. Coffee (COF) consumption increases antioxidant status and reduces oxidative stress. To determine whether this effect is due to its single or combined constituents, we studied the effect of COF, caffeine (CAF) and chlorogenic acid (CHLA) on food intake (FI), body weight (BW), body condition (BC), motor performance (MP), ability to move (AM), clinical score (CS), and antioxidant enzyme protein content (MnSOD; CAT; GR) in the brain of 108‐d old male G93A mice. Starting at age 40‐d, 21 mice were randomly divided into control (CON, 6), COF (5), CAF (5) or CHLA (5). Compounds were added to the food, equivalent to amounts found in 5‐10 cups of coffee/day (mg/g BW). FI increased over time (P < 0.001); all groups consumed more than CON (COF, 21%, P = 0.014; CAF, 22%, P = 0.018; CHLA, 12%, P = 0.086). BW increased and BC decreased over time (P < 0.001) with no differences between groups. MP increased over time (P < 0.001) and was higher in COF vs. all groups (CON, 6%; CAF, 27%; CHLA, 40%; NS). CAT was higher in COF vs. all groups (CON, 61%, P = 0.002; CAF, 25%, P = 0.028; CHLA, 22%, P = 0.023). There were no significant differences in CS, AM, MnSOD and GR. We conclude that COF, independent of CAF or CHLA, is protective in male G93A mice by improving motor performance and increasing antioxidant enzyme capacity.

Grant Funding Source

HHSF, NSERC, York U‐Health
Anti‐diabetic effects and action mechanism of Kochujang, a traditional fermented spicy paste, in type 2 diabetic animals
Authors: Park Sunmin , Kwon Dae Young , Hong Sang Mee , Ahn Il Sung
Published: June 2021

We examined the anti‐diabetic effects and action mechanism of Kochujang by investigating pancreatic β‐cell function and peripheral insulin resistance in 90% pancreatectomized (Px) diabetic rats fed high fat diets containing two different kinds of 5% Kochujang powder or the equivalent amount of nutrients for 8 weeks. They were made through the fermentation of two different kinds of meju (soybeans), red peppers, glutinous rice and malts. Meju was produced by fermenting soybeans in a traditional method (TMK) or in a more modern method in which soybeans are inoculated with Bacillus subtilus and Aspergilus sojae (MMK). TMK and MMK improved glucose tolerance by enhancing insulin sensitivity, but they did not potentiate glucose‐stimulated insulin secretion. The improvement in hepatic insulin sensitivity caused by TMK and MMK was explained by the potentiated phosphorylation of STAT3→AMPK→ACC and the reduced PEPCK expression. Kochujang diets reduced hepatic glucose output and triglyceride accumulation and increased glycogen storage. In conclusion, Kochujang improves glucose homeostasis by reducing insulin resistance, not by enhancing β‐cell function in diabetic rats. The improvement is associated with decreased hepatic fat storage via the activation of AMPK.
Vitamin D Therapy Reduces Blood Pressure: A Case Series
Authors: Judd Suzanne E , Raiser Sara , Kumari Meena , Tangpricha Vin
Published: June 2021

We investigated the effect of vitamin D repletion on resolution of hypertension in non‐whites using a pilot study. Subjects were recruited from the Atlanta Veterans Affairs Medical Center (VAMC) in Atlanta, GA. Subjects received a total dose of 600,000 IU D3 or placebo (n=3) taken weekly either over 3 weeks (n=2) or 12 weeks (n=3) or were given 0.5 ug calcitriol (n=2) taken twice daily for one week. We used a 24 hour ambulatory blood pressure (24 aBP) monitor to assess blood pressure. The calcitriol group experienced a 13% reduction in mean systolic aBP while the placebo group only decreased by 4% (p<0.001). One week after conclusion of calcitriol therapy 24 aBP pressure returned to pretreatment levels. In pooled analysis of all subjects randomized to vitamin D3, the mean 24 aBP decreased by 5% (p<0.001) in the group using D3. Compared with placebo, this change was not significant. Sub‐analysis demonstrated the individual experiencing the largest reduction in blood pressure using vitamin D3 had the highest plasma renin activity. Calcitriol had a strong rapid effect on blood pressure reduction while the effect of D3 may be limited to individuals with high plasma renin activity. Vitamin D therapy may be an effective intervention for reducing blood pressure.

Grant Funding Source

ASN and CF Foundation
Arsenic requires sphingosine‐1‐phosphate type 1 receptors to induce angiogenic genes and endothelial cell remodeling
Authors: Barchowsky Aaron , Klei Linda R , Straub Adam C , Stolz Donna B
Published: June 2021

Arsenic increases risk and incidence of cardiovascular diseases and cancers. Arsenic affects multiple vascular beds to promote liver sinusoidal capillarization and portal hypertension, ischemic heart disease, peripheral vascular disease, and tumor angiogenesis. The mechanism for initiating arsenic signaling in endothelial calls is unknown. We found that arsenic‐stimulated cell signaling and angiogenic gene expression in human microvascular endothelial cells (HMVEC) were Pertussis toxin sensitive indicating a G‐protein coupled signaling pathway. Both the sphingosine‐1‐phosphate type 1 receptor (S1P1) inhibitor VPC23019 and siRNA knockdown of S1P1 blocked arsenic‐stimulated HMVEC angiogenic gene expression and tube formation, but not induction of HMOX1or IL8. S1P1 was enriched on liver sinusoidal endothelial cells (LSEC) in vivo and VPC23019 inhibited arsenic‐stimulated oxidant generation and capillarization. These studies identified novel roles for S1P1 in mediating arsenic stimulation of both angiogenesis and pathogenic LSEC capillarization, as well as demonstrated a novel role for S1P1 in mediating environmental responses in the liver vasculature. Thus, they provide possible mechanistic insight into arsenic‐promoted vascular pathogenesis and disease promotion.
AlphaB crystallin regulation of ocular angiogenesis by modulation of vascular endothelial growth factor protein expression
Authors: Hinton David Ralph , Kase Satoru , He Shikun , Sonoda Shozo , Wawrousek Eric , Kannan Ram
Published: June 2021

AlphaB‐crystallin is a chaperone belonging to the small heat shock protein family. Herein we show attenuation of intraocular angiogenesis in αB‐crystallin knockout (αB(‐/‐)) mice in two models of intraocular disease: oxygen‐induced retinopathy and laser‐induced choroidal neovascularization. Retinal and choroidal neovascularization were prominently attenuated in αB(‐/‐) mice compared to wild‐type mice. Vascular endothelial growth factor‐A (VEGF) gene and hypoxia inducible factor‐1α expression were induced during retinal angiogenesis, but VEGF protein expression remained low in αB(‐/‐) retina vs wild‐type mice. Both αB‐crystallin and its phosphorylated form (pSer59) were expressed, and immunoprecipitation revealed αB‐crystallin binding to VEGF during angiogenesis in wild‐type mice. Cultured retinal pigment epithelial cells (RPE) derived from αB(‐/‐) mice showed low VEGF secretion compared to wild‐type cells. VEGF protein was ubiquitinated in human RPE after αB‐crystallin siRNA transfection and VEGF increased after proteasomal inhibition. Endothelial cell apoptosis in newly formed vessels was significantly greater in αB(‐/‐) than in wild‐type mice. Our studies point to the important role of αB‐crystallin in angiogenesis and its potential as a therapeutic target. Support provided by the Arnold and Mabel Beckman Foundation and NIH grants EY01545 and EY03040.
IP‐10 fragment is the functional motif that blocks endothelial cell motility and vessel formation
Authors: Yates Cecelia Chrsitina , Shields Danielle , Rodgers Margaret , Jaynes Jesse , Wells Alan , Bodnar Richard , Turner Timothy
Published: June 2021

Angiogenesis plays a critical role in optimum healing of a cutaneous wound. It requires well‐orchestrated integration of soluble and matrix factors and timely recognition of such signals to regulate this process. Previous work has shown that endothelial cells express CXC receptor 3 (CXCR3) late in wound regeneration and its ligand IP‐10 (CXCL10) which influences the cells ability to migrate and form vessels in vitro and in vivo resulting in inhibition and regression in the vascular network. To identify the functional domain of IP‐10 to develop new therapeutic agents to promote or inhibit blood vessel formation we isolated a 21 amino acid fragment, residues 78‐98, which we believe is the functional domain, of IP‐10 and analyzed its ability to modulate endothelial cell tube formation. Treatment of the endothelial cells with the IP‐10 fragment significantly inhibited VEGF‐induced endothelial motility and tube formation, a response critical for angiogenesis. Furthermore, simulation of CXCR3 by the IP‐10 fragments induced an increase in cAMP production and activation of PKA that leads to the inhibition of VEGF‐mediated m‐calpain activation and thereby limits cell motility. This activity is embodied in the minimal IP‐10 fragment. We posit that peptide design provides a novel system to provide desired functions of promoting and inhibiting signaling pathways and angiogenesis in human microvascular endothelial cells.
Parent‐specific inheritance of insulin resistance and NAFLD in AxB mice regulated by sex chromosomes
Authors: Rogers Arlin B. , Fox James G.
Published: June 2021

Nonalcoholic fatty liver disease (NAFLD) affects =30% of adults in the U.S., and is associated with obesity, metabolic syndrome, type II diabetes, and hepatocellular carcinoma (HCC). Here we report spontaneous insulin resistance, NAFLD and HCC with parent‐specific inheritance in AxB mice. Helicobacter‐free F1 offspring (n=59) from wild‐type C57BL/6 and A/JCr parents were maintained on a standard chow diet. ABF1 (A/J dam x B6 sire) but not BAF1 males (B6 dam x A/J sire) developed progressively severe hepatic steatosis and sporadic HCC by 15 months. NAFLD was a component of metabolic syndrome characterized by hyperinsulinemia, hypercholesterolemia and increased body mass (all P<0.05, unpaired t‐test). Liver microarray confirmed widely dysregulated metabolic signaling with immune activation. Single gene analysis of uniparentally expressed transcripts including mitochondrial, imprinted and sex‐linked genes revealed only a weak association with X‐linked thyroxine‐binding globulin. In contrast, phenotypic characterization of full‐length chromosome substitution (consomic) C57BL/6J‐Chr X/YA/NaJ mice showed a significant and additive contribution from both sex chromosomes. Our results demonstrate that parent‐specific inheritance of insulin resistance in AxB mice is regulated at the chromosomal level, and introduce a new model to study complex genetic determinants of NAFLD and HCC.
Morphogenetic Cell Movements – Beyond Epithelial‐to‐Mesenchymal Transition
Authors: Melani Mariana , Prasad Mohit , Jang Anna CC
Published: June 2021

The movements of cells that remain connected to one another in a group, known as collective cell movements, contribute critically to the morphogenesis of most organs and tissues. Such movements are remarkably diverse and cannot be explained by the well‐established phenomenon of epithelial to mesenchymal transition. We have been studying follicle cell movements in the Drosophila ovary using genetics and live‐imaging to identify the molecular mechanisms that generate diverse cell morphologies and arrangements. These studies together with others in the literature suggest that modular mechanical properties are used in a combinatorial manner to orchestrate morphogenesis.
Innate defense responses in Muc2 deficient mice: an important role for goblet cells
Authors: Burger‐van Paassen Nanda , Bouma Janneke , dez Bruijn Adrianus CJM , van der Sluis Maria , Van Seuningen Isabelle , Van Goudoever Johannes B , Renes Ingrid B
Published: June 2021

Background

Absence of a mucus layer, as in Muc2 knockout (KO) mice, leads the induction of colitis due to aberrant bacterial‐epithelial interactions. We hypothesize that innate defense molecules, i.e. bactericidal peptides, are upregulated in Muc2KO mice to compensate for loss of the mucus layer.

Aim

To study the innate defense response in Muc2KO mice.

Methods

Colonic tissue of Muc2KO and wild type (WT) mice was collected up to 28 days after birth. Expression of the bactericidal peptides; regenerating gene (Reg)3β, Reg3γ and angiogenin (Ang)4 was studied by qPCR, in situ hybridization and/or immunohistochemistry. Additionally, influx of T cells into the mucosa was studied as marker for inflammation.

Results

Compared to WT mice, Reg3β and Reg3γ mRNA levels were increased up to 50 fold in KO mice at 14 days, and a further increase was seen at 28 days. In contrast, Ang4 expression was observed in the colon of WT mice, but not in the colon of KO mice. Finally, the number of infiltrating T cells in the colon increased with age in KO mice.

Conclusions

Strong upregulation of Reg3β and Reg3γ in the colon of KO mice suggests increased innate defense capacity to compensate for the loss of intestinal barrier function. Reg3 gene expression correlates with the increased influx of T‐cells, implying a role for Reg3 peptides in intestinal inflammation. Absence of Ang4 expression in KO mice might indicate diminished bactericidal activity.
Interleukin‐1 receptor activates Rho kinase to disrupt epithelial tight junctions during Helicobacter pylori infection
Authors: Lapointe Tamia K , O'Connor Pamela M , Ménard Daniel , Buret Andre G
Published: June 2021

The ability of H. pylori (Hp) to modulate tight junction (TJ) permeability may contribute to the development of gastric ulcers and adenocarcinomas. Rho kinase (ROCK) and interleukin (IL)‐1β are known to modulate tight junction (TJ) function. While Hp induces IL‐1β expression, the role of this proinflammatory cytokine and ROCK in Hp‐mediated TJ disruption remains elusive.

AIM

This study investigated the role of ROCK and IL‐1β in Hp‐mediated TJ disruption in human gastric epithelial (HGE‐20) cells.

RESULTS

HGE‐20 monolayers challenged with either Hp or IL‐1β showed an increase in ROCK activity 6 h post infection, which coincided with redistribution and loss of claudin‐4. Inhibition of ROCK prior to bacterial challenge prevented these effects. Hp infection also induced IL‐1 receptor (IL‐1R) phosphorylation. Nevertheless, no increase in IL‐1β could be detected in supernatants of infected monolayers and treatment with co‐culture conditioned media failed to induce IL‐1R phosphorylation. While an IL‐1β neutralizing antibody did not prevent Hp‐induced IL‐1R activation, the use of an IL‐1R blocking antibody prior to infection prevented Hp‐mediated ROCK activation and claudin‐4 loss.

CONCLUSION

These findings suggest that the mechanism by which Hp disrupts gastric epithelial barrier structure involves IL‐1R ‐dependent activation of ROCK, which mediates claudin‐4 disruption. This work was funded by CIHR.
Tyrosine phosphorylation of VE‐Cadherin following activation of Src‐family kinases is not sufficient to decrease endothelial barrier function.
Authors: Adam Alejandro , Sharenko Amy , Vincent Peter
Published: June 2021

Src has been implicated as a major mediator leading to the loss of endothelial cell (EC) barrier function following exposure to inflammatory mediators and growth factors. One mechanism proposed for Src induced loss of endothelial barrier function is via phosphorylation of adherens junction proteins including VE‐cadherin. To assess the role of Src in EC barrier function independent of other signaling pathways, we activated Src directly either by expressing a constitutive active Src (caSrc) or by expressing dominant negative c‐Src kinase (CSK‐). Both strategies resulted in similar levels of Src activation as assessed by an increase in Src auto‐phosphorylation at Y419 and by phosphorylation of known Src substrates. Surprisingly, caSrc, but not CSK‐ expression, resulted in a loss of EC barrier function. Both caSrc and CSK‐ expression resulted in the phosphorylation of VE‐cadherin at Y658 and Y731. However, only CSK‐ induced the phosphorylation of Y685. Co‐IP and IF studies demonstrated that phosphorylation of VE‐cadherin did not impair VE‐Cadherin membrane localization nor its ability to bind p120 and β‐Catenin. CaSrc, but not of CSK‐, induced drastic changes in the cellular morphology, the actin cytoskeleton, focal adhesions and an increased MLC phosphorylation. Our data suggest that Src‐dependent phosphorylation of VE‐cadherin is not sufficient to decrease endothelial barrier function. Support R01HL77870
Pelvis has left the building: convergent evolution and development of stickleback fish
Authors: Shapiro Michael D
Published: June 2021

Convergent evolution is common among vertebrates, yet we understand comparatively little about the genetic basis of similar traits in different lineages. Ninespine and threespine stickleback fish provide many examples of convergent evolution of the skeleton and other traits, both within and between species. The genetic architecture of several adaptive traits is now known for threespine sticklebacks, and ninespine sticklebacks provide a unique opportunity to test whether similar or different chromosome regions underlie similar phenotypes in different lineages. We used quantitative trait locus mapping in ninespine sticklebacks to identify chromosome regions controlling pelvic (hind) fin development, lateral armor, and sex determination. Previous studies of threespine sticklebacks have implicated the repeated use of particular chromosome regions and genes in the evolution of similar phenotypes in different populations. In contrast, we found that pelvic loss, armor development, and sex determination in ninespine sticklebacks map to chromosome regions not previously known to control these traits. Hence, different regions of the genome mediate the evolution of similar traits in these two lineages.