bluuebunny/crossref_metadata_2025 huggingface dataset:
Colonic Inflammation Accompanies An Increase of β‐Catenin Signaling and Lachnospiraceae/Streptococcaceae in the Hind Gut of High‐Fat Diet‐Fed Mice
Authors: Zeng Huawei , Ishaq Suzanne L , Zhao Feng‐Qi , Wright Andre‐Denis G
Published: June 2021

Consumption of an obesigenic/high‐fat (HF) diet isassociated with an increase of inflammation‐related colon cancer risk and mayalter the gut microbiota. To test the hypothesis that a HF feeding accelerates inflammatory processes and changes gut microbiome composition, C57BL/6mice were fed a HF (45% energy) or low‐fat (LF) (10% energy) diet for 36 weeks. At the end of the study, body weights in the HF group were 35% greater than those in the LF group. These changes were associated with dramatic increases in inflammatory cell infiltration, inducible nitric oxide synthase (iNOS) protein concentration and cell proliferation marker (Ki67) in ileum and colon. Similarly, β‐catenin expression was increased in colon (but not ileum). Consistent with these gut inflammation phenotypes, we also found that plasma IL6 and tumor necrosisfactor‐α concentrations were also elevated in mice fed the HF diet, indicative of chronic inflammation. Fecal DNA was extracted and the V1–V3 hypervariable region of the microbial 16S rRNA gene amplified using primers suitable for 454‐pyrosequencing. Compared to the LH group, the HF group had high proportions of bacteria from the phylum Firmicutes, especially the family Lachnospiraceae which is known to be involved in the development of metabolic disorders, diabetes and obesity. Taken together, long‐term HF consumption not only increases inflammatory cell infiltration/molecular markers (e.g., iNOS), but also accompanies an increase of the Lachnospiraceae/Streptococcaceae in the hindgut of C57BL/6 mice.

Support or Funding Information

This work was supported by USDA‐ARS, CRIS project (5450‐51000‐050‐00D).
Maternal low protein diet leads to placental angiogenic compensation via dysregulated M1/M2 macrophages and TNFα expression in Sprague‐Dawley rats
Authors: Vomhof‐Dekrey Emilie E , Roemmich James N , Claycombe Kate J
Published: June 2021

A maternal low‐protein (LP) diet results in low birth weight, increased offspring rapid adipose tissue catch‐up growth, adult obesity, and insulin resistance in Sprague‐Dawley rats. The placenta plays key roles in nutrient transport and fetal growth. Placental function is dependent on regulation of immune cell populations and growth and angiogenic factors. Therefore, we hypothesize that a maternal LP diet leads to placental dysfunction through dysregulation of immune cell populations, cytokine production, and growth/angiogenic factors. Obese‐prone Sprague‐Dawley dams were fed 8% LP or20% normal protein diets for 3 weeks prior to breeding and through pregnancy. Maternal LP diet led to a decrease in placental weight and efficiency. LP diet placent as had an increase in angiogenic factors, FGF2, VEGFR‐1, IGF2, M2macrophages producing TNFα and a decrease in M1 macrophages and iNKT cells. These results suggest that prenatal protein restriction forces the placenta to upregulate compensating mechanisms of angiogenesis in order to fulfill the nutrient demands of the fetus.

Support or Funding Information

This work was supported by USDA Agricultural Research Service Project #3062‐51000‐052‐00D.
Effect of High Folic Acid Diet on Mitochondrial DNA Content in Young Adult and Aged Mice
Authors: Rakic Jelena Mustra , Sawaengsri Hathairat , Reginaldo Christina , Selhub Jacob , Paul Ligi
Published: June 2021

Mitochondrial DNA (mtDNA) codes for proteins of oxidative phosphorylation system and maintaining adequate mtDNA copy number is necessary for cell survival. Studies in animal models have shown that folate deficiency can affect mtDNA content. Current data suggests excess folic acid intake can result in conditions that resemble folate deficiency. We tested the hypothesis that excess folic acid in diet can influence mtDNA gene content in old and young mice. Young (3 months) and old mice (16 months) mice were fed a control (1xRDA) or high folic acid (20 x RDA) diet for 3 months (n=8–11/ group). The colonic mucosal mtDNA content was determined by real‐time PCR using primers specific for mtDNA and normalized to an endogenous reference gene, mapK. The data were analyzed using student's t‐test. Among mice on control diet, mtDNA content of old mice was 0.4 fold that of the young mice (p=0.004). There was no difference in mtDNA content of young mice on control vs high folic acid diet. MtDNA content of the old mice fed a high folic acid diet was 7‐fold higher than those fed a control diet (p=0.001). While higher mtDNA copy number is considered to have beneficial effects on survival, very high mtDNA copy number has been shown to be accompanied by nucleoid enlargement, defective transcription and mtDNA deletions in old mice. Thus the dramatic increase in mtDNA content due to excess folic acid diet may be detrimental. Further research is required to establish if the high mtDNA content due a high folic acid diet can result in mitochondrial dysfunction in aged mice.

Support or Funding Information

Support from USDA cooperative agreement 51520‐008‐04S and pilot grant from JM USDA HNRCA on Aging at Tufts University
Lower hepatic iron storage associated with obesity in mice can be restored by decreasing body fat mass through feeding a low fat diet
Authors: Chung Hak , Wu Dayong , Smith Donald , Meydani Simin Nikbin , Han Sung Nim
Published: June 2021

Previously, we showed that high fat diet (HFD)‐induced obesity resulted in lower hepatic iron storage. To further investigate the effect of diet‐induced increase in adiposity on hepatic iron storage level and determine whether these obesity‐related changes could be reversed by decreasing fat mass through feeding a low fat diet, we compared hepatic iron levels in HFD‐induced obese mice after their diets were switched to a low fat diet with those continued to be fed HFD. Five‐week‐old female C57BL/6 mice were assigned to three groups – LL group was fed control diet containing 10% kcal fat for 31 weeks, HH group was fed HFD containing 60% kcal fat for 31 weeks, and HL group was fed HFD for the first 15 weeks and switched to the control diet for the following 16 weeks. Fat mass and lean body mass were measured by MRI at 14th and 30th weeks. To assess the hepatic iron storage, total iron content was measured by ICP‐MS and ferritin protein levels were determined by Western blot analysis. mRNA levels of hepatic hepcidin (Hamp) (the key factor which regulates body iron level) and hepatic and duodenal BMP‐6 (Bmp6) (a signaling molecule known to affect hepcidin expression) were determined by real time PCR. Body weights were significantly different among groups (35.8 ± 1.9 g, 40.6 ± 1.3 g, 63.1 ± 1.5 g in LL, HL, and HH groups, respectively). Fat mass was significantly different among groups (11.4 ± 2.0 g, 15.8 ± 1.0 g, 37.5 ± 1.1 g in LL, HL, and HH groups, respectively). Fat mass of HL group decreased from 19.0 ± 1.1 g at 14th wk to 15.8 ± 1.0 g at 30th wk. Total hepatic iron content of HL group was similar to that of LL group, which was about 30% higher than that of HH group (74.2 ± 7.5 μg/g tissue, 72.7 ± 2.6 μg/g tissue, 55.7 ± 3.8 μg/g tissue in LL, HL, and HH groups, respectively, p<0.05). Liver ferritin protein levels were similar in LL and HL groups, and significantly lower in HH group. These measures of iron status were significantly negatively correlated with body fat mass (r=−0.627, p<0.01 for liver iron content, r=−0.687, p<0.001 for ferritin). Liver Hamp mRNA level was not statistically different among groups. Liver Bmp6 expression was not significantly different among groups. In duodenum, however, Bmp6 mRNA level was significantly lower in HH group than LL and HL groups (p<0.01). Duodenal Bmp6 mRNA levels showed positive correlation with hepatic iron content (r=0.564, p<0.05) and hepatic Hamp expression (r=0.517, p<0.05). In summary, hepatic iron storage level is closely related to body adiposity. Reducing body fat mass through feeding a lower fat diet to HFD‐induced obese mice restores liver iron storage by lowering duodenal Bmp6 mRNA levels.

Support or Funding Information

Supported by research funds through the Research Institute of Human Ecology (RIHE) in Seoul National University and USDA contract #58‐1950‐0‐014.
In Vivo Suppression of Atherosclerosis by Dietary Oats Avenanthramides
Authors: Thomas Michael , Kim Sharon , Collins F. William , Wise Mitchell L. , Meydani Mohsen
Published: June 2021

The consumption of oatmeal and oat bran has been shown to reduce total and low density lipoprotein (LDL) cholesterol levels in plasma, which are major risk factors for coronary heart disease (CHD). Oats, in addition to containing soluble fiber (β‐glucan), are a good source of protein and lipids as well as several antioxidants including vitamin E, phytic acid, phenolics, and Avenanthramides (Avns), unique soluble bioactive compounds present only in oats. Avns can inhibit inflammation in arterial endothelium by suppressing NF‐κB, which regulates expression of several inflammatory cytokines. We have reported that Avns suppressed endothelial expression of chemokines (MCP‐1, IL‐8), pro‐inflammatory cytokines (IL‐1 and IL‐6) and several adhesion molecules, all of which suggest oats’ potential anti‐inflammatory and anti‐atherogenic properties. In agreement with our in vitro observations, supplementing a high fat diet of LDLr−/− mice with feed containing 27% or 40% oats significantly reduced aortic lesion development. However, this effect of an oat‐based diet cannot be attributed only to its cholesterol lowering effect; rather, it is due to the additive/synergistic anti‐inflammatory interaction with oat Avns. To elucidate Avns’ contribution to the inhibition of atherogenesis, we supplemented a high fat diet of LDLr−/− mice with regular oats or false‐malted oats for 16 wks. Avns’ concentration as measured by LC/MS‐MS in the regular oat diet was 10 ppm; in the false‐malted oat diet, it was 451 ppm. Food intake was not different between the groups, but high fat‐fed mice gained body weight significantly (p<0.05). Both oat‐based diets (malted or false‐malted) significantly (p<0.05) reduced high fat diet‐induced atheroma lesions in the aortic tricuspid valve, but there was no difference in the extent of lesions between these two groups. However, LDLr−/− mice fed a high fat diet containing false‐malted oats with high levels of Avns had significantly (p<0.05) lower numbers of lesions in the descending aorta than mice fed a low fat diet, a high fat diet, or a high fat diet supplemented with regular oats. A non‐significant trend toward reduction of expression of VCAM‐1 in the lesions of aortic valves of mice fed high fat diet containing high levels of Avns was observed. Importantly, reduction of blood total cholesterol levels was the same in both oat‐supplemented groups, suggesting that false‐malted oats, with their higher concentrations of Avns, contributed more to the reduction of aortic lesions. These preliminary in vivo data suggest that consuming oats, especially false‐malted oats, may be beneficial in the prevention of cardiovascular disease.

Support or Funding Information

Supported by USDA‐ARS agreement #58‐1950‐0‐014
Effects of Extra Virgin Olive Oil (EVOO) Oleocanthal and Oleacein Content on Platelet Reactivity in Healthy Adults
Authors: Agrawal Karan A , Holt Roberta R , Li Xuequi Shirley , Melliou Eleni , Pedersen Theresa Lynn , Wang Selina C , Flynn Dan , Magiatis Prokopios , Newman John W.
Published: June 2021

Data from limited dietary intervention trials suggest that the cardiovascular health benefit of EVOOs may increase as their phenolic antioxidants content increases. However, while EVOOs contain an array of bioactive compounds, little information exists regarding the physiological effects of specific chemical species. Among the EVOO‐derived phenolics with demonstrated anti‐inflammatory and antioxidant effects in animal and in vitro models is oleocanthal, an inhibitor of cyclooxygenase (COX). The current study compared the impact of acute intake (40 mL) of EVOO on platelet reactivity in healthy adult males (n=9). Three EVOOs were characterized and chosen for equivalency in their total phenolic content and fatty acid profiles, but differing in their oleocanthal to oleacein ratio. In a randomized double‐blind cross‐over study design, subjects consumed EVOOs that provided either a ~2:1 oleocanthal/oleacein (Oleo+) or 2:1 oleacein compared to oleocanthal (Olea+). A third EVOO served as a control, providing minimal amounts of oleocanthal and oleacein but had a similar total phenolic content in the form of free tyrosol (Oleo− Olea−). A positive control for to platelet response was obtained after the intake of 400 mg of ibuprofen on a fourth study visit. Blood samples for optical platelet aggregometry were collected at baseline, and 2 hours post EVOO intake; with samples for oxylipin analysis collected from the activated platelet supernatants. Maximum platelet aggregation (Pmax) was significantly reduced 2 hours after Ibuprofen intake compared to all EVOOs when platelets were activated with either 1 μg/mL or 3 μg/mL of collagen. No significant differences between EVOOs for Pmax were observed with 3 μg/mL of collagen. However, scaling each individuals’ response to their range of responses on the 4 treatments, 1 μg/mL collagen‐induced Pmax was significantly reduced (repeated measures ANOVA, p<0.05) for Oleo+, but not Olea+ compared to Oleo− Olea− 2 hours after intake (7.33 ± 7.9, −12.4 ± 12, and −29.3 ± 11, mean ± SEM percent basal activity, respectively). These changes in Pmax were strongly correlated with the change in the sum of COX and 12‐lipoxygenase (LOX) (r2 = 0.53), although considerable individual variability in both platelet response and COX and LOX product formation was noted. Taken together the data suggests that platelet response after EVOO intake is dependent on the presence of specific phenolics.

Support or Funding Information

This project was supported by funds from the Captain Vassilis Foundation, GAEA Products SA, and USDA Intramural project 2032‐51530‐022‐00D. The USDA is an equal opportunity provider and employeer.
Butyrate Modulates Cav‐1 and Its Binding Partner AhR, Leading to Differential Cyp1a1 and Cyp1b1 Gene Expression in Vascular Endothelial Cells
Authors: Hoffman Jessie B. , Petriello Michael C , Hennig Bernhard
Published: June 2021

Dietary fiber and associated metabolites such as butyrate may protect against vascular inflammation and atherosclerosis. The vascular endothelium plays an important role in the overall inflammatory response and early pathology of atherosclerosis. Membrane microdomains called caveolae and their functional proteins, e.g., caveolin‐1 (Cav‐1), are involved in the regulation of many inflammatory pathways, particularly in endothelial cells. Caveolae sequester multiple proteins at the lipid membrane that promote a microenvironment for efficient inflammatory responses. Emerging data now implicate an interactive role between caveolae, xenobiotic detoxification, and oxidative stress. Past literature has shown that downregulation of Cav‐1 can prevent the toxicity of environmental pollutants. The aryl hydrocarbon receptor (AhR) regulates the expression of xenobiotic metabolizing enzymes or cytochrome P450s, such as Cyp1a1 and Cyp1b1, which have been linked to vascular inflammation. We hypothesize that fiber‐derived butyrate is protective by modulating caveolae‐linked regulatory genes, including AhR and subsequent Cyp1a1 expression. Primary endothelial cells were treated with increasing concentrations of butyrate for up to 8 hours and subsequently examined for expression of AhR, Cav‐1, Cyp1a1, Cyp1b1 and antioxidant enzymes. Our preliminary data show that 4 h treatment with physiologically relevant concentrations of butyrate significantly decreased Cav‐1 and AhR mRNA levels. Also, the AhR targets Cyp1A1 and Cyp1B1 were drastically reduced. Our laboratory has previously demonstrated that up‐regulation of Cav‐1, AhR and relevant CYP enzymes by polychlorinated biphenyls (PCBs) induces inflammation, which can be blocked by silencing Cav‐1. Thus, our data suggest that dietary fiber‐derived metabolites or diets enriched in fiber have the potential to protect against environmental insults and associated inflammatory events.

Support or Funding Information

Supported by grants from NIEHS, NIH (P42ES007380), and US Department of Agriculture, UK AES; Graduate stipend provided by by NIH Training Grant T32 DK007778.
Lactobacillus rhamnosus and Flavanol‐enriched Cocoa Powder Altered the Immune Response to Infection with the Parasitic Nematode Ascaris suum in a Pig Model
Authors: Jang Saebyeol , Lakshman Sukla , Molokin Aleksey , Urban Joseph F. , Davis Cindy D , Solano‐Aguilar Gloria
Published: June 2021

Immunomodulatory effects of the probiotic bacteria Lactobacillus rhamnosus (LGG) and prebiotic effects of flavanol‐enriched cocoa have been previously reported. This study evaluated if feeding LGG and flavanol‐enriched cocoa powder to pigs would modulate the host response against a prototypical type 2 allergic immune response induced by infection with Ascaris suum. Three month old male pigs (n=8/diet) were fed diets containing 1 x 1010 cfu LGG/day or placebo (maltodextrin) and 0 or 26 grams of flavanol‐enriched cocoa powder (Acticoa, Barry‐Callebaut, Belgium)/day for 7 weeks in a 2 x2 design. Seventeen days before euthanasia, all pigs were inoculated with infective Ascaris suum eggs to mimic type 2 allergic immune response. LGG abundance in feces and intestinal contents, expression of immunomodulatory genes in the tracheobronchial lymph nodes (TBLNs), alveolar macrophages (AM), and mesenteric lymph nodes (MLNs) were determined by RT‐PCR. When compared to placebo‐treated pigs, the abundance of LGG was increased in the intestinal contents and feces of pigs fed LGG. Consumption of cocoa powder together with LGG did not affect LGG abundance. Ascaris suum induced interleukin‐13, eosinophil peroxidase, and chemokine ligand 26 gene expression levels were reduced in TBLNs of pigs fed LGG regardless of cocoa consumption. LGG consumption induced an increase in toll‐like receptor (TLR)‐9 and tumor necrosis factor (TNF)‐α gene expression in AM of infected pigs. In contrast, TLR2 gene expression in AM was only increased in response to feeding cocoa powder. After 24 hr ex vivo stimulation with LPS, AM from LGG and cocoa powder fed pigs had increased TNFα gene expression compared with other treatment groups suggesting a synergistic effect in response to the infection. Moreover, LGG fed pigs exhibited a significant increase of TLR2 and TLR4 in MLNs compared to placebo‐treated pigs. Overall, this study demonstrated that feeding LGG reduced Ascaris suum‐induced eosinophil activity in TBLNs and increased gene expression of TLRs and pro‐inflammatory TNF‐α, while consumption of cocoa powder alone modulated TLR2 expression in AM. The combination of feeding LGG and cocoa powder synergistically stimulated TNFα gene expression in LPS‐stimulated AM.

Support or Funding Information

This study was supported by USDA Project Number 8040‐51530‐056‐00 and Inter agency agreement 8040‐51530‐056‐13 with ODS at NIH
Changes in the Phytochemicals Composition during Bread and Pancake Preparations from Whole and Refined Wheat Flours
Authors: Luthria Devanand L , Lu Yingjian , Fuerst E. Patrick , Morris Craig F.
Published: June 2021

In this study, we investigated the changes in the level of phytochemicals during bread and pancake preparations from refined and whole‐wheat flours of two wheat varieties. Phytochemicals were extracted with ultrasonic (UAE) and microwave (MAE) assisted extractions followed by analyses with spectroscopic and chromatographic methods. Comparison of the efficacy of two methods for hydrolysis and extraction of phenolic acids showed that yield of total phenolic acids (TPA) was ~10–25% higher among all varieties using UAE as compared to MAE. Most (> 90%) of the phenolic acids in all wheat flour samples existed in insoluble bound form. Ferulic acid, lutein and α‐tocopherol were the predominant phenolic acid, carotenoid, and tocopherol, respectively, extracted from all fractions. The quantities of phenolic acids, carotenoids, and tocopherols were significantly higher in all fractions made from whole wheat flour than their corresponding refined wheat flour fractions. Ferulic acid was the most abundant phenolic acid, accounting for a mean of 85% and 90% of TPA among all RF (refined flour) and WW (whole wheat) samples, respectively. The concentration of TPA did not significantly changed during pancake and bread preparation. However, concentration of tocopherols and carotenoids was reduced. Thus the processing of wheat samples has varying effects on the concentration of phytochemicals in raw and finished products. This project was partially supported by the Agriculture and Food Research Initiative Grant no. 2009‐02347 from the USDA National Institute of Food and Agriculture.

Support or Funding Information

This project was partially supported by the Agriculture and Food Research Initiative Grant no. 2009‐02347 from the USDA National Institute of Food and Agriculture.
The Effectiveness of Coenzyme Q1 and Q10 in Mitigating Myocardial Reperfusion/Ischemia (MI/R) Injury
Authors: Aahitey Myles , Sutton Jakob , Venditto Joseph , Kucharski Kevin , Robbie Jason , Kuo Daniel , Barsotti Robert , Young Lindon , Chen Qian
Published: June 2021

Mitochondria may be a principle source of oxidative stress causing MI/R injury. Coenzyme Q10 (CoQ10) is essential for electron transport in normal mitochondria, has antioxidant properties but its bioavailability is likely reduced due to oxidative stress during MI/R. Coenzyme Q1 (CoQ1) is a derivative of CoQ10, but is a more potent antioxidant than CoQ10 due to a shorter isoprene chain. We hypothesize that CoQ1 will exhibit better cardioprotective effects during MI/R. CoQ1 (MW=250 g/mol; 20 μM, n=4) and CoQ10 (MW=863 g/mol; 20 μM, n=5) were given at reperfusion in isolated rat hearts subjected to I (30 min)/R (45 min). We found that MI/R hearts (n=7) and MI/R+DMSO hearts (n=4) (1% DMSO was used to solubilize CoQ1 and CoQ10) exhibited significantly compromised cardiac contractile/diastolic pressures and coronary flow during reperfusion compared to those of sham hearts (n=5). By contrast, the final left ventricular developed pressure was significantly improved by CoQ1 treatment (53.5±5.5 mmHg), but not CoQ10 treatment (38.4±8.6 mmHg), when compared to that in MI/R hearts (33.6±6.2 mmHg) and MI/R+DMSO hearts (36.4±9.7 mmHg) (p<0.05). Similarly, the final peak of the first derivative of left ventricular pressure was significantly higher in CoQ1 treatment (1180.2±103.0 mmHg/s), but not CoQ10 treatment (770.6±120.1 mmHg/s), when compared to that in MI/R hearts (700.6±134.7 mmHg/s) and MI/R+DMSO hearts (and 741.5±168.6 mmHg/s) (p<0.05). CoQ1 and CoQ10 treated hearts showed no improvement on diastolic pressure and coronary flow compared to the controls. Moreover, infarct size was reduced by CoQ1 treatment (25±3%) and CoQ10 treatment (29±4%) compared to that in untreated MI/R (44±6%) and MI/R+DMSO (32±4%). In summary, our preliminary results indicate that CoQ1 was more effective than CoQ10 in restoring post‐reperfused cardiac contractile function, but not infarct size during MI/R.

Support or Funding Information

This study was supported by Division of Research, the Center for Chronic Disorders of Aging, and Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.
Plant Alkaloid Desensitization of Fetal Muscle‐Type Nicotinic Acetylcholine Receptors
Authors: Green Benedict , Lee Stephen , Welch Kevin , Cook Daniel
Published: June 2021

The exposure of a developing fetus to certain plant alkaloids has the potential to inhibit fetal movement which can then result in developmental defects. The putative mechanism of movement inhibition is the desensitization of fetal muscle‐type nicotinic acetylcholine receptors (nAChR) which is affected by agonist chemical structure. In this study, we tested the hypothesis that a 1,2‐dehydropiperidine alkaloid agonist is more effective at desensitizing nAChR than a saturated analogue. Agonists were evaluated in TE‐671 cells which express fetal muscle‐type nAChR, and SHSY‐5Y cells which express autonomic‐type nAChRs using a membrane potential sensing dye system. Anabaseine, a 1,2‐dehydropiperidine alkaloid, was more effective at desensitizing TE‐671 cells than anabasine which is fully saturated at the 1,2 position. The neurotransmitter acetylcholine which was included as a control was more effective than the pyridine alkaloid nicotine at desensitizing the agonist response in TE‐671 cells. Both nicotine and acetylcholine were equally effective at desensitizing the agonist response in SH‐SY5Y cells. These results suggest that anabaseine, a 1,2‐dehydropiperidine alkaloid agonist is more effective at desensitizing fetal muscle‐type nAChR than anabasine and has a greater potential to inhibit fetal movement.

Support or Funding Information

This research was supported by USDA/ARS.
Temperature‐Oxygen Interactions and the Evolution of Giant Antarctic Sea Spiders
Authors: Woods H. Arthur , Moran Amy L. , Tobalske Bret W , Lane Steven J , Shishido Caitlin L.
Published: June 2021

For more than a hundred years, scientists have known that many marine invertebrate taxa grow to unusually large size in polar waters. This biogeographic pattern has become known as polar gigantism, and it occurs in taxa as diverse as glass sponges, ctenophores, foraminiferans, polychaete annelids, isopod crustaceans, copepods, amphipod crustaceans, and pycnogonids. Despite being common, polar gigantism remains a physiological and evolutionary mystery; how and why do polar taxa grow to such large sizes? Using Antarctic sea spiders (pycnogonids), we tested one of the leading physiological hypotheses about polar gigantism—the oxygen hypothesis, proposed by Chapelle and Peck, which states that polar giants are permitted in very cold water by high levels of oxygen supply coupled to low metabolic demand for that oxygen. We measured whole‐body metabolic rates spanning three orders of magnitude in body size. Metabolic rate scaled to body mass with an exponent of 0.9, indicating that giant sea spiders do not require unusually low metabolic rates. Using oxygen electrodes and dye‐tracer experiments, we examined the details of oxygen transport across the cuticle and of oxygen levels and transport in the hemocoel. Oxygen is taken up across the cuticle (there is no distinct respiratory organ), and the cuticle is a much greater barrier to oxygen movement than is the mix of fluids and tissues inside the legs and trunk. Oxygen levels in the hemocoel were high everywhere in the body, but were consistently highest in distal parts of the legs and lowest in the trunk. In addition, larger sea spiders had lower central levels of oxygen. Our dye tracer experiments suggested that contractions of the heart and muscles in the legs drive relatively rapid blood circulation, especially between the trunk and proximal parts of the legs. This indicates that sea spiders use their proximal leg segments as respiratory surfaces. Building on these physiological details, we constructed a mathematical model of oxygen transport and consumption, which we applied to the range of body sizes we studied in Antarctica. The model accurately predicted measured levels of oxygen in the blood. We then used the model along with realistic Q10s for key oxygen processes to simulate how polar sea spiders would perform in warmer water. These simulations strongly suggest that gigantic forms would not obtain oxygen rapidly enough in warm water to support their higher metabolic rates. Thus, together our empirical and theoretical studies support the oxygen hypothesis: it appears that the giant forms found in the Ross Sea can balance their oxygen budgets only because supply is so high and demand is so low.

Support or Funding Information

National Science Foundation grant 1341476
Heart Rate Regulation in the Killer Whale
Authors: Bickett Natalie , Tift Michael , St. Leger Judy , Ponganis Paul
Published: June 2021

Heart rate responses and gas exchange ultimately underlie the diving capacity/foraging ecology of cetaceans, and may well contribute to the morbidity/mortality of whales stranded after exposure to naval sonar. In order to examine cardiac responses in mid‐sized whales, and eventually measure cetacean heart rates in the wild, we applied a digital electrocardiogram (ECG) recorder with a suction cup attachment technique to a 3868 kg killer whale, Orcinus orca. Heart rate during 10‐min spontaneous, voluntary rest periods ( both apneic and eupneic heart rate) ranged from 21 to 82 beats min−1 between eupnea and apnea, was variable during apnea, and averaged 50 ± 3 SD beats min−1, 1.6 × the resting heart rate predicted by Stahl's allometric equation. During trained, stationary submersions of two to three‐min duration, and during one‐min duration, leisurely underwater swims at 0.5 Hz fluke stroke rate, heart rate was 34 ± 5 SD, and 59 ± 4 SD, respectively. Large oscillations in heart rate with rapid return to baseline occasionally occurred under all conditions. The “resting” heart rate of this killer whale was appropriate for its body mass, but probably elevated due to study conditions (non‐fasting). Sub‐surface swimming elicited an increase in heart rate, and voluntary, trained submersions resulted in a dive response with a variable bradycardia. This approach shows promise to further investigate heart regulation in mid‐sized cetaceans and eventually record heart rates of free‐diving whales.

Support or Funding Information

ONR Award No N000141410404
An Optogenetic Test for the Functional Specificity of Bulbospinal Sympathoexcitatory Neurons
Authors: Pracejus Natasha H , Farmer David GS , McAllen Robin M
Published: June 2021

Sympathetic preganglionic neurons are functionally specialised, each belonging to one of a limited number of functional classes (e.g. muscle vasoconstrictor, cutaneous vasoconstrictor, sudomotor). How far this functional specificity applies to the premotor neurons that drive them is uncertain. We devised an optogenetic test for the existence of premotor neurons that are collateralised to drive more than one functional class of sympathetic neuron.

Bulbospinal neurons were transduced by a retrogradely transported viral vector (canine adenovirus) encoding Cre recombinase (CAV‐CMV‐Cre; provided by Eric Kremer, Montpellier). This was injected around sympathetic preganglionic neurons at the T12‐L1 spinal levels. At the same time, a Cre‐activated adeno‐associated virus (AAV) encoding Channelrhodopsin‐2 (ChR2) and the fluorescent label mCherry (AAV1.EF1.dflox.hChR2(H134R)‐mCherry.WPRE.hGH; provided by Deisseroth, Stanford via UPenn) was injected into the left RVLM. Only neurons transduced by both viral vectors should then express the ChR2 and mCherry. Spinal injections were made at T12‐L1 because they were considered unlikely to overlap with the preganglionic supply to the heart. The bulbospinal presynaptic terminals infected with CAV‐Cre were thus presumably on preganglionic neurons that supplied end organs such as hindlimb muscle, but not the heart.

Terminal functional experiments under urethane anaesthesia were performed 2–8 weeks after the viral injections. Activity was simultaneously recorded from the left cardiac and lumbar sympathetic nerves, whilst pulses of blue laser light (473 nm, 10–12 mW, 20 ms) were flashed onto the left RVLM through a 100 μm diameter fibre‐optic probe.

In about one‐third of experiments, we were able to show functional activation of sympathetic nerves that was time‐locked to the flashed laser light. In all successful experiments, we found cardiac sympathetic activation as well as lumbar sympathetic activation (peak response latencies 135–150 ms and 215–230 ms, respectively). Subsequent fluorescence microscopy on brainstem sections confirmed the presence of small numbers of mCherry expressing bulbospinal neurons in those rats.

This study demonstrates that at least some bulbospinal neurons send excitatory synaptic inputs to preganglionic neurons of more than one functional type. It also demonstrates the feasibility of using anatomically‐based optogenetic transduction as a test for functional collateralisation in central pathways.

Support or Funding Information

Australian Research Council DP13014661
Long‐term Leucine and BCAA Inclusion in a 30% Protein and Energy Restricted Diet Increases mTORC1 Signaling in Skeletal Muscle of Neonatal Pigs
Authors: Manjarin Rodrigo , Columbus Daniel A , Solis Jessica , Suryawan Agus , Hernández‐García Adriana D , Nguyen Hanh V , Fiorotto Marta L , Davis Teresa
Published: June 2021

Suboptimal nutrient intake during the shift from parenteral to oral feeding represents a limiting factor for growth of low‐birth weight infants. Leucine has been shown to have anabolic properties in skeletal muscle and can promote lean gain when supplemented to neonatal pigs fed a milk replacement formula. The objective of this study was to test if enteral leucine supplementation alone, or in combination with isoleucine and valine, can increase muscle protein synthesis in neonate pigs who consume 30% less crude protein (CP) and metabolizable energy than their requirements, and whether this effect is mediated through changes in muscle protein synthesis and degradation pathways, and amino acid (AA) transporter expression. Piglets (4‐d‐old) were fed by gastric tube 1 of 4 diets providing (kg BW/d) 15 g CP and 215 kcal (CON), 10.5 g CP and 156 kcal (R), 10.5 g CP + 1.19 g leucine and 156 kcal (RL), or 10.5 g CP + 1.67 g branched‐chain amino acids and 156 kcal (RBCAA) at 4‐h intervals for 9 d (n = 24). Body weights (BW) were recorded on d 0, 3, 6, and 9. On d 9, plasma AA and insulin concentrations and muscle protein synthesis rate, abundance of Na+‐coupled neutral AA transporter (SNAT2) and L‐type AA transporter (LAT1), and phosphorylation and/or abundance of insulin receptor substrate 1 (IRS‐1, Ser1101), protein kinase B (PKB), S6 kinase (S6K1), 4E‐binding protein (4EBP1), eukaryotic initiation factor 4 complex (eIF4E·eIF4G), forkhead box O (FOXO1, Ser256), atrophy F‐Box/Atrogin‐1 and muscle RING‐finger protein (MuRF1) were determined post‐feeding. Plasma leucine levels were greater in RL and RBCAA compared to CON (P ≤ 0.01). BW gain and muscle protein synthesis rate, phosphorylation of S6K1 and 4EBP1, and formation of eIF4E·eIF4G were lower in R, RL, and RBCAA than CON (P < 0.01). RBCAA and RL upregulated (P ≤ 0.01) S6K1 and 4EBP1 phosphorylation and eIF4E·eIF4G formation compared to R. Phosphorylation of FOXO1 was lower in R compared to CON (P ≤ 0.05). There were no differences in phosphorylation and/or abundance of IRS‐1, PKB, SNAT2, LAT1, atrogin‐1 and MURF1 among diets. In conclusion, when protein and energy intakes are restricted, leucine or BCAA supplementation for 9 d increases the activation of the mammalian target of rapamycin pathway, but does not enhance skeletal muscle protein synthesis or promote body weight gain in neonatal pigs. Furthermore, protein degradation signaling, AA transporter abundance, and insulin signaling are unaffected by leucine or BCAA supplementation of a protein and energy restricted diet suggesting that when both dietary protein and/or energy supplies are deficient, the anabolic effect of leucine or BCAA supplementation on muscle protein accretion is limited.

Support or Funding Information

NIH AR444474, NIH HD072891, NIH AR46308, USDA NIFA 2013‐67015‐20438, and USDA/ARS 6250‐51000‐055
White Blood Cell Counts during Lower Body Negative Pressure vs. Blood Loss in Humans
Authors: van Helmond Noud , Johnson Blair D , Curry Timothy B , Cap Andrew P , Convertino Victor A , Joyner Michael J
Published: June 2021

Hypovolemia‐induced sympathoexcitation might initiate an immune response through an increase in circulating epinephrine and splenic demargination. We tested the hypothesis that lower body negative pressure (LBNP) and blood loss (BL) result in comparable increases in white blood cell counts (WBCs). We determined WBCs and epinephrine levels in 12 men who performed a LBNP and BL protocol in a randomized order. LBNP consisted of 5‐minute stages at 0, ‐15, ‐30, and ‐45 mmHg of suction. BL included 5 minutes at baseline and following three stages of 333 mL of blood removal (up to 1000 mL total). Blood draws were performed at baseline and after the last stage of each protocol. LBNP to −45mmHg was a greater central hypovolemic stimulus vs. BL, therefore WBCs and epinephrine concentrations were plotted against central venous pressure (CVP) to obtain stimulus‐response relationships using the linear regression line slopes for both protocols. Paired tests were used to determine if the slopes of these regression lines fell on similar trajectories for each protocol. Mean regression line slopes fell on similar trajectories for total WBC (LBNP 183±46 vs. BL 155±109 mcL−1 *mmHg−1, p=0.15), neutrophils (LBNP 110±29 vs. BL 96±72 mcL−1 *mmHg−1, p=0.15) and lymphocytes (LBNP 65±21 vs. BL 59±38 mcL−1 *mmHg−1, p=0.90). Mean regression line slopes for epinephrine were similar as well (LBNP 15±5 vs. BL 16±4 pg *mL−1 *mmHg−1, p=0.84). Our results indicate that LBNP models the stimulus‐response relationship between CVP and WBCs observed during BL. Central hypovolemia, induced by LBNP or BL, elicits a relative leukocytosis with a predominantly neutrophil‐based response.

Support or Funding Information

US Army MRMC Combat Casualty Care Research Program Grant # W81XWH‐11‐1‐0823
Hemodynamic responses to simulated hemorrhage: Role for the carotid bodies
Authors: Holbein Walter W , Johnson Blair D , Convertino Victor A , Curry Timothy B , Joyner Michael J
Published: June 2021

Background

Lower body negative pressure (LBNP) increases carotid body chemosensitivity. Whether the increase in chemosensitivity is functionally significant in the hemodynamic response to LBNP was previously unknown. We tested the hypothesis that desensitization of the carotid body chemoreceptors would improve the hemodynamic response to LBNP.

Methods

Ten healthy, non‐obese male subjects (35 ± 3 years, BMI: 25 ± 1 kg/m2) completed 2 study visits (Saline, Dopamine), separated by a minimum of 4 weeks. Intravenous dopamine was infused at an individualized dose (1–2 mg/kg/min) shown to maximally blunt the hypoxic ventilatory response (a measure of carotid body chemosensitivity). Arterial blood pressure (brachial catheter) and heart rate (ECG) were continuously recorded. On each study day subjects underwent step‐wise exposures of LBNP (−15, −30, and −45 mmHg) to simulate hemorrhage. Cumulative stress index (CSI) was calculated by summating the product of negative pressure (mmHg) by the time (min) during each LBNP level (−15, −30, and −45 mmHg).

Results

Dopamine tended to reduce CSI (Saline: 503 ± 52; Dopamine: 408 ± 45 mmHg*min; p=0.07). Changes in heart rate from baseline during graded LBNP were not statistically distinguishable between Saline and Dopamine conditions (−15 mmHg: +4 ± 1 vs +3 ± 2 BPM, p=0.99; −30 mmHg: +12 ± 1 vs +12 ± 3 BPM, p=0.99; −45 mmHg: HR +21 ± 2 vs +19 ± 5 BPM, p=0.99). Similarly, change in systolic blood pressure from baseline during graded LBNP was not different between Saline and Dopamine conditions (−15 mmHg: +1 ± 1 vs −1 ± 2 mmHg p=0.99; −30 mmHg: −7 ± 3 vs −6 ± 4 mmHg, p=0.99; −45 mmHg: −12 ± 4 vs −22 ± 6 mmHg, p=0.19). However, change in diastolic blood pressure from baseline during graded LBNP was higher at −45 mmHg between Saline and Dopamine conditions (−15 mmHg: +2 ± 1 vs −0 ± 1 mmHg p=0.47; −30 mmHg: −1 ± 2 vs −2 ± 2 mmHg p=0.26; −45 mmHg: −2 ± 2 vs −12 ±3 mmHg, p=0.0004).

Conclusions

Desensitization of the carotid body chemoreceptors reduced diastolic blood pressure responses to LBNP at −45 mmHg only, with minimal effect at lower levels. These data suggest chemoreflex activity during LBNP may be functionally significant in the blood pressure responses only during higher levels of simulated hemorrhage.

Support or Funding Information

Funding: W81XWH‐13‐2‐0038 (DOD)
Reduced Short‐chain Fatty Acid Producing Microbiota are Linked to Increased Energy Intake and Susceptibility to High Fat Diet Induced Hepatic Steatosis in Low Aerobic Capacity Rats
Authors: Panasevich Matthew R , Morris E M , Chintapalli S V , Wankhade U , Shankar K , Britton S L , Koch L G , Thyfault J P , Rector R S
Published: June 2021

Poor aerobic fitness is linked to nonalcoholic fatty liver disease and increased all‐cause mortality. We have previously shown that low aerobically fit, low capacity running (LCR) rats fed an acute high fat diet (HFD; 45% kcal from fat) for 3 days displayed a greater positive energy balance due to a greater increase in energy intake and reduced energy expenditure, which likely played a role in their susceptibility for hepatic steatosis compared with high aerobically fit, high capacity running (HCR) rats. Here, we tested the hypothesis that poor physiological outcomes in LCR rats following acute HFD feeding are associated with alterations in cecal microbiota. Through amplification of the V4 region of bacterial 16S rDNA, we found that at the phylum level, the LCR rats had a reduced relative abundance of cecal Proteobacteria (P<0.05) and increased Cyanobacteria (P<0.05) compared with HCR rats. Interestingly within Proteobacteria, Desulfovibrionaceae, and unassigned genera within Desulfovibrionaceae, were increased (P<0.05) in HCR compared with LCR rats, suggesting increased sulfur reduction from endogenous substrates (i.e. mucins and proteins). Conversely, the Proteobacteria family Alcaligenaceae was increased (P<0.05) in LCR compared with HCR rats, which may be related to susceptibility to NAFLD in the LCR rats. While acute HFD feeding reduced the overall relative abundance of Firmicutes in both strains, short‐chain fatty acid (SCFA)‐producing Phascolarctobacterium and Butyricimonas were reduced (P<0.05) only in the LCR rats. In addition, SCFA‐producing Ruminococcae (r=−0.75) and Ruminococcus (r=−0.73, P<0.01) were negatively correlated with energy intake only in the LCR rats. Overall, these data suggest that the susceptibility to acute HFD‐induced increases in energy intake and hepatic steatosis in low aerobically fit animals is associated with changes in the cecal microbiome, particularly suppression of SCFA‐producing bacteria.

Support or Funding Information

AHA 14POST20110034 (EMM), NIH R01DK088940 (JPT), P40OD021331 and R01DK099034 (LGK and SLB), USDA‐CRIS 6206‐51000‐007‐00D‐05 (KS), VA‐CDA2 IK2BX001299 (RSR).
The NADPH‐Production Enzyme Isocitrate Dehydrogenase Maintains Quiescence in Hair Follicle Stem Cells
Authors: Ambrus Aaron Michael , Lemons Johanna , Raitman Irene , Krishnan Nithya , Jelinek David , Suh Eric , Remillard Matthew , Shilo Noya , Haley Erin , Sun Lova , Xiao Roy , Flores Aimee , Wang David , White Andrew , Lowry William , Coller Hilary
Published: June 2021

NADPH is a critical metabolite that is important for regenerating reduced glutathione from oxidized glutathione and eliminating reactive oxygen species (ROS). Metabolic flux and microarray experiments in fibroblasts demonstrated that NADPH‐producing enzymes, glucose‐6‐phosphate dehydrogenase (G6PD), isocitrate dehydrogenase (IDH), and the upstream transcription factor NRF2, are all activated in quiescent compared with proliferating fibroblasts. We demonstrated that G6PD and NRF2 are functionally important for quiescence by showing that inhibition of G6PD or NRF2 results in oxidative stress and apoptosis specifically in quiescent fibroblasts. Flow cytometry experiments demonstrated that ROS in quiescent cells can be derived from mitochondrial superoxide that results from increased mitochondrial activity in the serum‐starved compared with proliferating fibroblasts, as well as an increase in reactive nitrogen species that arise from peroxisomes. To understand the physiological role of NADPH‐production pathways, we examined their potential activity in mouse skin. Consistent with our findings in quiescent fibroblasts, in situ metabolic activity assays revealed higher potential activity for G6PD and IDH in non‐dividing cells. Of particular interest was the high IDH potential in quiescent hair follicle stem cells. Staining of live mouse skin with monochlorobimane showed higher reactivity, consistent with higher levels of reduced glutathione, in hair follicle stem cells. Inhibition of IDH activity in healthy mouse skin with two different inhibitors resulted in progression in the hair follicle cycle from a quiescent to proliferative state suggesting that the high IDH activity observed in hair follicle stem cells may contribute to the maintenance of these stem cells in a quiescent state. Going forward, our focus is on understanding the role of IDH in the maintenance of stem cells as opposed to proliferating, committed progenitor cells.

Support or Funding Information

H.A.C. was the Milton E. Cassel scholar of the Rita Allen Foundation. This work was supported by NIGMS Center of Excellence grant P50 GM071508, two grants from the Cancer Institute of New Jersey, the New Jersey Commission on Cancer Research, National Cancer Institute 1RC1 CA147961‐01, a Focused Funding Grant to H.A.C. from the Johnson & Johnson Foundation and a grant from the PhRMA Foundation to H.A.C, and XXXX to W.E.L. J.M.S. was supported by NIH training grant T32 HG003284. E.M.H. acknowledges a Bowen Fellowship from Princeton University and the New Jersey Commission on Cancer Research. E.J.S. acknowledges support from the National Science Foundation.
Subacute neuroendocrine and behavioral effects of underwater trauma and predator exposure in rats
Authors: Moore Nicole L.T. , Cravedi Kevin , Vuong Chau , Sousa Jason C , Marcsisin Sean R
Published: June 2021

Traumatic stress physiological response and recovery encompasses a wide range of measurable behavioral and neuroendocrine changes in rats. Acute stress response and return to baseline may vary across differing neuroendocrine signaling molecules and behavioral manifestations, and may also be influenced by extrinsic factors such as the specific stressor presented or phase of the circadian cycle during which responses are evaluated. While rodent models of traumatic stress have been used to evaluate acute and chronic changes in these measures, few longitudinal studies have been done to characterize multiple interrelated neuroendocrine markers across the circadian cycle and compare to acute and lasting behavioral changes. We set out to describe the subacute effects of traumatic stress exposure on exploratory behavior, acoustic startle response, and a panel of neuroendocrine markers across the circadian cycle. Rats were exposed to either underwater trauma (UWT) or predator exposure (PRED). Then repeated behavioral tests (exploratory behavior on the elevated plus maze, acoustic startle) were conducted across 7 days, or in yoked cohorts, blood samples were collected at 10 time points over the next 7 days. Neuroendocrine markers were quantified from serum using LC‐MS or ELISA, analyzed over time, and compared against behavioral responses at several corresponding time points. Preliminary data show differential responses for UWT and PRED across the experimental timeline, and distinct response timelines for the different measures collected.

Support or Funding Information

Supported by the Military Operational Medicine Research Program, US Army Medical Research and Materiel Command.