bluuebunny/crossref_metadata_2025 huggingface dataset:
Consumption of up to Three Eggs per Day Increases Dietary Cholesterol and Choline while Plasma LDL Cholesterol and Trimethylamine N‐oxide Concentrations Are Not Increased in a Young, Healthy Population
Authors: Lemos Bruno S , DiMarco Diana M , Missimer Amanda , Murillo Ana Gabriela , Malysheva Olga V , Caudill Marie A , Blesso Christopher N , Fernandez Maria Luz
Published: November 2023

Eggs are a source of many nutrients, including cholesterol and choline, thus there is uncertainty regarding the impact of egg intake on plasma lipids and the choline metabolite trimethylamine‐N‐oxide (TMAO). In particular, it has not been established whether there is a dose‐dependency between egg consumption and changes in plasma lipids and plasma TMAO concentrations. Therefore, the effects of increasing daily egg intake on biomarkers associated with cardiovascular disease (CVD) risk were evaluated in a young, healthy population. Thirty‐eight subjects (19 men/19 women, 24.1 ± 2.2 y, BMI 24.3 ± 2.5 kg/m2) were recruited for this 14‐week crossover intervention. Participants underwent a 2‐week washout period with no egg consumption (0 eggs/d), followed by the intake of 1, 2, and 3 eggs/d for 4 weeks each. Anthropometric data (weight, body mass index (BMI), waist circumference (WC)), blood pressure (BP), dietary records, and plasma biomarkers (lipids, glucose, liver enzymes, C‐reactive protein (CRP), choline, and TMAO) were assessed following each study period. BMI, WC, systolic BP, plasma glucose, triglycerides, total cholesterol, liver enzymes, and CRP were unchanged during the intervention. Diastolic BP decreased with increasing egg intake (p < 0.05). Intake of nutrients present in eggs (fat, cholesterol, choline, selenium, and vitamin D) increased with egg intake (p < 0.05) while carbohydrate and fiber intake and glycemic load decreased (p < 0.05). Compared to 0 eggs/d, intake of 1 egg/d increased HDL cholesterol (HDL‐c) (p < 0.05); HDL‐c remained elevated with intake of 2–3 eggs/d. Compared to 0 eggs, Intake of 1 egg/d resulted in a 10.9% decrease in plasma LDL cholesterol (LDL‐c) (p < 0.05) while there was no difference in LDL‐c between intake of 0, 2, or 3 eggs/d. Intake of 1–3 eggs/d also resulted in a decreased LDL‐c/HDL‐c ratio (p < 0.01) when compared to 0 eggs/d. Lastly, plasma choline increased (p < 0.0001) in a dose‐dependent manner with egg intake but remained within established normal ranges, while fasting plasma TMAO did not change. These data indicate that intake of up to 3 eggs/d results in an overall beneficial effect on biomarkers associated with CVD risk in a healthy population, as documented by increased HDL‐c and plasma choline and reductions in the LDL‐c/HDL‐c ratio, in combination with unchanged plasma LDL‐c and TMAO concentrations.

Support or Funding Information

This research was funded by an award to MLF by the Esperance Family Foundation and a grant awarded to DMD by the Egg Nutrition Center.
A 12‐week Randomised Controlled Trial to Evaluate Effects of Dietary Pulse Consumption on Cardiovascular Disease Risk Factors
Authors: Hill Alison M , Zahradka Peter , Coates Alison M , Howe Peter RC , Taylor Carla G , Buckley Jon D
Published: November 2023

Pulses are rich in soluble fiber, protein and polyunsaturated fatty acids, and low in sodium and saturated fat. Some studies have reported that pulse (or legume) consumption improves lipid and glucose control, and there is emerging evidence that pulses may improve vascular health. The aim of the present study was to determine whether modest increases in pulse consumption could improve traditional cardiovascular (CV) disease risk factors in older, overweight adults. This study was a multi‐site, 12 week randomized controlled trial conducted in Australia (n=74) and Canada (n=66). One hundred and forty adults (63.0 ± 7.2 years, BMI 31.5 ± 4.0 kg/m2) who were habitually low pulse consumers (i.e. ≤ ½ cup of pulses per week) were randomized to incorporate commercially available pulse‐enriched foods [~100g/day (i.e. ¾ cup/day) pulses] or energy‐matched control foods into their usual diet for 12 weeks. Body weight, blood pressure, arterial stiffness, lipids, glucose, insulin and C‐reactive protein were measured at baseline, 6 and 12 weeks. Five‐day weighed food records captured dietary data at baseline and Week 12 (data currently available for Australian cohort only). Pulse intake increased significantly in the Pulse vs Control group by week 12 (109 ± 39g vs. 2 ± 11g, respectively P<0.001). CV risk factors did not change in either group by Week 12. However, there was a significant inverse relationship between changes in pulse intake (g) and changes in plasma triglyceride levels (r = −0.23, P=0.036) after controlling for changes in carbohydrate intake. Data from the Australian Cohort suggest a dose‐response effect of pulse consumption on circulating triglycerides. Larger intakes of pulses may be required to facilitate changes in other CV risk factors. Our data indicate that the health effects associated with incorporating higher levels of pulses into the diet can be effectively obtained by employing canned pulses.

Support or Funding Information

Grain Research Development Corporation, Department of Further Education, Employment, Science and Technology (DFEEST) and Manitoba Science, Energy & Technology (International Collaboration Fund)
Substitution Of Carbohydrate With Whole Eggs Or Egg Whites Attenuates Postprandial Hyperglycemia‐Mediated Impairments In Vascular Endothelial Function In Prediabetic Men By Limiting Lipid Peroxidation And Dysregulated Arginine Metabolism
Authors: McDonald Joshua D , Chitchumroonchokchai Chureeporn , Li Jinhui , Volek Jeff S , Ballard Kevin D , Bruno Richard S
Published: November 2023

Eggs attenuate postprandial hyperglycemia (PPH), a predictor of cardiovascular disease‐related mortality. We hypothesized that acute ingestion of egg‐based meals with a glucose challenge would protect against PPH‐mediated impairments in vascular endothelial function (VEF) by attenuating lipid peroxidation and dysregulated arginine metabolism. A randomized, cross‐over study was conducted in prediabetic men (n = 20) who ingested isocaloric meals (400 kcal) containing 100 g glucose (GLU), or 75 g glucose with 1.5 whole eggs (EGG), 7 egg whites (WHITE), or 2 egg yolks (YOLK). Brachial artery flow‐mediated dilation (FMD), plasma glucose, insulin, malondialdehyde (MDA), inflammatory proteins, and arginine (ARG) and its related metabolites [asymmetric dimethylarginine (ADMA); symmetric dimethylarginine (SDMA); homoarginine (hARG)] that regulate nitric oxide (NO•) biosynthesis were assessed to calculate postprandial AUC0–180 min. Compared with GLU, FMDAUC was higher (P<0.01) in EGG and WHITE only. Glucose, insulin, and MDA AUCs were lower (P<0.05) in EGG and WHITE only. Compared to GLU, ARGAUC was similarly higher (P<0.01) in EGG and YOLK, but highest in WHITE. AUCs of ADMA/ARG, SDMA/ARG, and hARG/ARG were lower (P<0.01) in EGG and YOLK, and lowest in WHITE. GlucoseAUC correlated with MDAAUC (r = 0.36; P<0.01), and each correlated with ADMA/ARGAUC (r = 0.29–0.34; P<0.05) whereas only glucoseAUC correlated with SDMA/ARG (r = 0.29, P<0.05). Inflammatory proteins (C‐reactive protein, myeloperoxidase, TNFα) were unaffected (P>0.05) by all egg meals. Substituting carbohydrate with whole eggs, likely through its egg white fraction, protects against impairments in VEF in prediabetic men independent of inflammation by reducing PPH that otherwise increases lipid peroxidation and alters arginine availability for NO• synthesis by nitric oxide synthase.

Support or Funding Information

Supported by American Egg Board/Egg Nutrition Center.
The role of milk fat globule membranes in behavior and cognitive function using a suckling rat pup supplementation model
Authors: Brink Lauren Roukia , Lonnerdal Bo
Published: November 2023

Milk fat globule membranes (MFGM) surround droplets delivering lipids to the breast‐fed infant and are enriched with glycoproteins upon exocytosis out of the mammary cell. Its diverse array of proteins provided by the lipid bilayer of the mammary epithelial cells, along with the lipid compounds it contains, is thought to play an influential role in infant development. MFGM is typically removed during processing of cow milk for the generation of infant formula. Recent clinical trials have found that formula supplemented with bovine MFGM improved cognition. Here, we aimed to determine the mechanisms behind this improved developmental outcome. Sprague Dawley rats were bred and their litters were culled to either 10 pups to represent normal growth (N) or 16 pups to represent restricted growth (R) per dam. From postnatal day (PD) 2, pups were supplemented daily by oral gavage with MFGM or whey protein (control) at 100 mg/kg body weight. Pups were supplemented until PD13 and killed or supplemented until PD21 and then later exposed to cognitive testing (Tmaze and Passive avoidance). The R whey protein supplemented animals had lower Tmaze scores than the N whey protein animals. The R MFGM supplemented animals, however, did not show a decrease in cognitive scores. Restricted growth affected the passive avoidance test scores as well; however, an effect of the MFGM supplement was not seen. At PD14, MFGM was shown to increase mRNA expression of genes involved in brain function in both N & R animals, including BDNF (1.25‐fold change (fc) N, 1.48 fc R), St8sia4 (1.67 fc N) and Glutamate Receptor‐1 (1.71 fc N, 1.54 fc R) (P<0.01). Our findings suggest that MFGM plays an influential role in cognitive development by up‐regulating proteins involved in brain function.

Support or Funding Information

Arla Foods
Conceptualizations of Nutrition and Food Choice Behaviors Among Adolescent Girls and Young Women in Harare, Zimbabwe
Authors: Tinago Chiwoneso Beverley , Ingram Lucy Annang , Frongillo Edward A. , Blake Christine E. , Engelsmann Barbara , Simmons David
Published: November 2023

Objective

The latest Zimbabwe National Nutrition Strategy highlights that malnutrition is one of the most serious health problems affecting women of child‐bearing age in Zimbabwe. Furthermore, adolescence is a critical phase for improving nutrition status, yet little is understood about adolescent girls' and young women's perspectives on nutrition, a key component to aid in improving nutritional status. The research study took an emic approach to understand and describe how adolescent girls and young women (14–24 years) in Harare, Zimbabwe conceptualize nutrition and how these conceptualizations inform their food choice behaviors.

Methods

Semi‐structured, in‐depth, qualitative interviews were conducted in both Shona and English with adolescent girls and young women aged 14–24 years (N=48) who lived in two low‐income high‐density communities in Harare. Data were analyzed thematically using NVivo 10 software.

Results

Nutrition was mainly conceptualized in terms of the characteristics and utility of food with adolescent girls and young women in the study conceptualizing nutrition across four themes; health, nutritious food (Chikafu chinovaka muviri ‐ Food that builds the body), affordability and availability of nutritious food, and food that fills you up. Participants aged 14–20 years primarily conceptualized nutrition in terms of a balanced diet, while those aged 21–24 years emphasized health and the affordability and availability of nutritious food. Most participants identified traditional home‐cooked meals such as sadza (maize staple food), rice with peanut butter, fruits, pumpkin leaves, and wild vegetables as the main sources of nutrition. The affordability and availability of nutritious food also impacted participants' meal planning. Although most participants aged 14–20 years described that other family members mostly decided what they ate, they were the ones who cooked most of the family meals. Among participants who chose what to cook, they described how planning for meals occurred daily and this was dependent on what food was available in the home. Most also described how, besides the readily stocked staple foods like sadza, they purchased additional food ingredients daily.

Conclusions

Future approaches to improve nutritional status among adolescent girls and young women in Zimbabwe should acknowledge the socio‐cultural views concerning nutrition and target social networks and the additional structural environmental influences on food choice behaviors. Study findings will be used to inform approaches to strengthen efforts to improve the nutritional status of adolescent girls and young women in Zimbabwe.

Support or Funding Information

This research was funded by the Sackler Institute for Nutrition Science at the New York Academy of Sciences.
Folic Acid Content in Gestational Diet Elicits Dose‐dependent Changes on Hypothalamic Feeding Circuits in Wistar Rats at Birth
Authors: Yang Neil Victor , Chatterjee Diptendu , Pannia Emanuela , Kubant Ruslan , Anderson Gerald Harvey
Published: November 2023

Background

Folate plays an important role in early brain development of offspring, notably on neural stem cell proliferation and differentiation. Excess (10‐fold) intakes of folic acid (FA) in the gestational diet have been linked to increased food intake and obesity in adult offspring. Hypothalamic neuronal populations play a critical role in regulating food intake and maintaining energy balance.

Objectives

To identify neuronal populations susceptible to FA intakes during pregnancy, we measured the expression and maturation of neuropeptide Y (NPY+) and proopiomelanocortin (POMC+) containing neurons, known to induce opposing effects on food intake regulation. Furthermore, we tested if these outcomes could be explained by altered folate metabolism in the brain, which could lead to epigenetic reprogramming of neuronal differentiation and maturation.

Methods and Design

Pregnant Wistar rats were assigned to one of 5 AIN‐93G diets (n=12/group) providing FA at: 0, 1.0 (control), 2.5, 5.0, or 10.0‐fold of recommended level. Expression of NPY and POMC neuronal populations were assessed in coronal hypothalamic sections from male rat offspring, collected at birth. Immunohistochemical staining for the neuronal nuclear antigen (NeuN) measured neuron quantity and maturation. Hypothalamic levels of folate metabolites were assessed by HPLC analysis.

Results

A dose‐dependent effect (P <0.05) was identified between higher intakes of gestational FA and increased expression of mature NPY‐containing neurons in the hypothalamus, at birth. Pups from 2.5‐ and 5.0‐fold FA groups had 2.2 and 3.1‐fold more mature NPY‐neurons than control, respectively. However, gestational FA content had no effect on the number and maturation of POMC‐expressing neurons. Deficient (0‐fold) and high (10.0‐fold) FA gestational diets lead to higher levels of tetrahydrofolate (THF, 2.4 fold, P <0.0001) and 5‐methyltetrahydrofolate (MTHF, 3–4 fold, P <0.0001) in the hypothalamus, compared to control.

Conclusion

FA content in the gestational diet elicits a dose‐dependent effect on the number of mature hypothalamic NPY neurons in male Wistar rats at birth, possibly associated with greater accumulation of folate metabolites in the brain. This indicates that hypothalamic food intake pathways are sensitive to the dose of gestational FA and its bioactive metabolites, which may influence metabolic phenotype of offspring later in life.

Support or Funding Information

Canadian Institute of Health Research, Institute of Nutrition, Metabolism and Diabetes (CIHR‐INMD), Reference MOP‐130286.
Racial/ethnic disparities in childhood body mass index trajectories and potential causes among 29,254 US children: the Early Childhood Longitudinal Study‐Birth and Kindergarten Cohorts
Authors: Wang Youfa , Min Jungwon , Wen Xiaozhong , Xue Hong
Published: November 2023

Objective

Describe recent sex‐ and ethnic‐specific childhood BMI growth trajectories of US children, and explore the potential causes of racial/ethnic disparities in childhood BMI trajectories, including household socio‐economic status (SES) and family rules for children's TV watching, bedtime, and meal time.

Methods

The sex‐ and ethnic‐specific children's mean BMI trajectory curves between 7 months to 16 years of age were fitted by mixed effect models with fractional polynomial functions using 29,254 children from two US nationally representative cohorts, Early Childhood Longitudinal Study Birth (ECLS‐B, 2001) and Kindergarten (ECLS‐K, 1998–99). Multivariate regression model was used to examine the mediating roles of household SES and family rules on racial/ethnic disparities in childhood BMI trajectory characteristics.

Results

Hispanic boys (HB) and African American girls (AAG) continuously had the highest mean BMI after adiposity rebound than their same sex counterparts. They had the earliest adiposity rebound (mean [SD]: HB‐57.9 [7.8]; AAG‐59.0 [7.2] months), steeper growth velocity (HB‐5.7 [1.8]; AAG‐7.0 [1.5] 10−2 kg/m2/month), and highest area under curve (HB‐2724.5 [489.8]; AAG‐2681.2 [426.7] kg/m2*month) from adiposity rebound to 16 years of age. The racial/ethnic disparities in childhood BMI trajectories were partially mediated by household SES and family rules for children's regular bedtime (p<0.05).

Conclusions

In the US, racial/ethnic disparities in childhood BMI trajectories are apparent starting from adiposity rebound. Hispanic boys and African American girls have unfavorably high BMI trajectories. These disparities can be substantially explained by household SES level and family rules for children's bedtime. Efforts to reduce obesity disparities in different races/ethnicities should start early and focus on preventing early life risk factors related to sociodemographic characteristics and family rules for children's daily routine.

Support or Funding Information

NIH, 1R01HD064685‐01A1
Loss of a Healthy Weight Advantage among Foreign‐born Children of Mexican Origin: An Analysis of Overweight/Obesity Trends from 1988–1994 to 2007–2014
Authors: Maldonado Luis E. , Albrecht Sandra S.
Published: November 2023

We evaluated whether foreign‐born Mexican‐American (MA) children continued to exhibit a health weight advantage in recent years relative to U.S.‐born MA children given Mexico's emerging obesity epidemic. We also investigated the implications of these changing patterns on overweight/obesity disparities between MAs and non‐Hispanic whites (NHW) over time. Using data from MA and NHW children ages 5–17 years participating in the National Health and Nutrition Examination Surveys (1988–1994 (N=4,022) and 2007–2014 (N=4,713), we calculated odds ratios (OR) of overweight/obesity by ethnicity‐nativity (US‐born MA, foreign‐born MA, and NHW), adjusting for socio‐demographic covariates, and an interaction between ethnicity‐nativity and survey period. In 1988–1994, foreign‐born MAs had significantly lower adjusted odds of overweight/obesity compared to US‐born MAs (OR = 0.53, 95% CI: 0.33, 0.85). However, by 2007–2014, the nativity gap in overweight/obesity closed (OR = 0.85; 95% CI: 0.65, 1.11), indicating that the healthy weight advantage once held by foreign‐born MAs was no longer evident in recent years. Moreover, although predicted probabilities estimated from logistic regression models suggested foreign‐born MAs had the lowest predicted probability of overweight/obesity (25%) in 1988–1994 compared to both US‐born MAs (39%) and NHW children (32%), by 2007–2014, foreign‐born and US‐born MAs had comparable estimates (60% and 64%, respectively), both higher than for NHW's (57%). The healthy weight advantage previously held by foreign‐born MA children appears to have eroded in recent years. Although disparities in childhood overweight/obesity between MAs and NHWs had previously been specific to US‐born MAs, disparities in recent years have extended to foreign‐born MAs. Continued monitoring of overweight/obesity trends in foreign‐born MA children, as well as those born in the US, is critical given the long‐term impacts on cardio‐metabolic outcomes, which already disproportionately burden Mexican‐Americans adults.

Support or Funding Information

NIH/NHLBI (1‐T32‐HL129969‐01A1) and the Carolina Population Center (R24 HD050924)
Evaluation of ‘JenMe’, a Commercially‐Delivered Weight Management Program for Adolescents: a Randomised Controlled Trial
Authors: Bonham Maxine P , Dordevic Aimee L , Ware Robert S , Truby Helen
Published: November 2023

Objective

Adolescent obesity is difficult to treat and widely accessible treatment options with proven efficacy remain scarce. This study evaluated a commercially available, structured short‐term weight management program designed for obese adolescents delivered by non‐health professionals.

Methods

A multi‐site parallel‐group randomised controlled trial was conducted to evaluate a commercial 12 week lifestyle behavioral program in commercial weight management centres in Australia. Eligible participants (13–17 years, BMI z score ≥1.282 with no presenting morbidities) were randomized (n=88) to intervention or wait‐list and the program was delivered by consultants at participating weight management centres. The primary outcome was change in BMI z score. Secondary outcomes included the psychometric variables quality of life, body esteem and self‐esteem. Data was analyzed according to intention‐to‐treat principles.

Results

Of 88 participants randomised, 74 enrolled into the program and 12‐week data was available for 55 individuals (74%). A significantly greater decrease in BMI z‐score in the intervention group (n=32) was observed when compared with the wait‐list control group (mean difference (MD) = −0.27 kg/m2; 95% confidence interval [CI], −0.37, −0.17; P<0.001). Participants allocated to receive the lifestyle intervention reported a greater improvement in body esteem (MD=2.3, 95%CI, 0.6, 4.1; P=0.009) and quality of life (MD=7.8, 95%CI, 1.6, 14.0; P=0.01) compared with the wait‐list control group. Similarly, intervention group participants had greater physical activity levels (MD=0.09 metabolic equivalents, 95%CI, 0.02, 0.17; P=0.03) compared with the wait‐list control group.

Conclusions

This evaluation provides evidence that a structured lifestyle intervention delivered by a commercial provider in an adolescent population can result in clinically relevant weight loss and improvements in psychosocial outcomes in the short term. Further research is required to evaluate long term outcomes

Support or Funding Information

The study was funded by Jenny Craig Weight Loss Centres Pty Ltd as a research grant to A/Prof Bonham and Prof Truby. By contractual agreement, scientists (MB, HT, AD) at Monash University and other participating institutions (RW) have responsibility and independence regarding data management, analysis and publication.
Untargeted Metabolomics Reveal Disparate Metabolite Profile in Follicular Fluid between Obese and Normal Weight Women
Authors: Ruebel Meghan L , Piccolo Brian D , Moutos Dean M , Shankar Kartik , Andres Aline
Published: November 2023

The microenvironment of follicular fluid is thought to provide a rich source of nutrients and other factors that promotes oocyte growth and viability; however, it is not known if obesity or related sequelae impacts this environment. To address this research gap, we designed a clinical study to assess follicular fluid metabolite profiles, inflammatory markers and oocyte gene expression. Normal weight women (NW; n=13) and overweight/obese (OW; n=11) undergoing fertility treatments were recruited prior to an oocyte retrieval procedure. Anthropometrics, body composition (Air Displacement Plethysmography), and overnight fasting serum samples were collected. During the oocyte retrieval procedures, follicular fluid aspirates containing the oocyte samples were also collected. Leptin, C‐reactive protein, triglycerides, and total cholesterol were assessed in serum and follicular fluid using colorimetric and ELISA assays. Gas chromatography time of flight‐mass spectrometry was used to characterize the metabolome of serum and follicular fluid. Discriminant metabolites of NW and OW samples were identified using a backwards iterative selection strategy using ranked variable importance in projection measurements from partial least squared‐discriminant analysis (PLS‐DA) models. By design, OW women had significantly higher BMI, fat mass, and serum HOMA‐IR compared to NW women (p<0.01). Leptin and C‐reactive protein concentrations were increased in both serum (5.3±1.0 vs. 27.2±6.6 and 3.2±0.7 vs. 5.7±1.0, respectively) and follicular fluid (11.1±2.4 vs. 26.8±6.7 and 2.7±0.8 vs. 5.9±1.8, respectively) from OW women and follicular fluid triglyceride (25.8±14 vs. 64.2±46.9, respectively) and total cholesterol (21.1±3.2 vs. 36.3±7.6, respectively) levels were elevated in OW women compared to NW (p<0.05) women. Metabolites selected as discriminant of NW and OW follicular fluid samples by PLS‐DA included elevated uric acid, isothreonic acid, 2‐deoxytetronic acid and 4 unknown metabolites in OW women. Conversely, 2‐dimethylacetal‐ketoglucose, aminomalonate, and 2 unknown metabolites were significantly decreased in OW relative to NW women. Other serum metabolites discriminant of NW and OW serum included cholesterol, mannose, palmitic acid, sucrose, and 5 unknown metabolites. These results suggest that the follicular fluid metabolome differs between NW and OW women and may potentially contribute to altered oocyte metabolism and development. One unknown metabolite, BB112556, was significantly lower in both follicular fluid and serum, raising the possibility of a serum biomarker of oocyte health and development. Combined with previous findings of differential oocyte gene expression in NW vs. OW women from this cohort, these results suggest that obesity significantly affects oocyte transcription and follicular fluid metabolites.

Support or Funding Information

ACHRI CUMG 035716 and USDA□ARS Project 6026□51000□010□05S
Nicotinamide Riboside, an NAD+ Precursor, Attenuates The Activation of Hepatic Stellate Cells
Authors: Pham Tho X. , Bae Minkyung , Lee Ji‐Young
Published: November 2023

Recent evidence suggests that supplementation of nicotinamide riboside (NR), a natural NAD+ precursor found in milk, can reduce liver fibrosis in several mouse models of non‐alcoholic steatohepatitis. However, the molecular mechanism by which NR reduces liver fibrosis remains to be elucidated. Hepatic stellate cells (HSCs) are primarily responsible for the production and deposition of collagen and other extracellular matrix (ECM) substances in the liver during injury. Upon liver injury, HSCs become activated and transdifferentiate into a‐smooth muscle actin (aSMA)‐positive myofibroblasts, which produce ECM proteins such as collagen 1a1 (COL1a1). Therefore, we determined whether the preventive effect of NR on liver fibrosis is attributed to its effect on the activation of HSCs. LX‐2 cells, a human HSC cell line, were pretreated with or without 500 mM of NR for 24 h followed by treatment of 4 ng/mL of transforming growth factor β1 (TGFβ1), a potent pro‐fibrogenic cytokine, for an additional 24 h in the absence or presence of NR. TGFβ1 markedly increased the protein levels of aSMA and COL1a1, while NR pretreatment significantly attenuated the TGFβ1‐induced increase in COL1a1 protein levels. Despite the reduction in COL1a1 protein by NR, its mRNA levels were significantly increased by NR, suggesting that NR may regulate COL1a1 expression at the post‐transcriptional level in LX‐2 cells. We next isolated mouse primary HSCs from C57BL/6 mice and activated them by culturing on a plastic dish for 7 days in the absence or presence of 500 μM NR. Compared to mouse primary HSCs cultured for 2 days, which maintain their quiescent state, HSCs activated for 7 days expressed significantly higher levels of aSMA and COL1a1 mRNA. When NR was added to HSCs during the activation for 7 days, aSMA and COL1a1 mRNA levels were significantly lowered when compared to the cells that were activated in the absence of NR. This suggests that NR treatment attenuated the activation of quiescent mouse primary HSCs to myofibrolasts. Together, we demonstrated that treatment of NR attenuated HSC activation in human and mouse models of HSCs, which suggests that anti‐fibrotic action of NR in the liver may be attributable, at least in part, to its inhibitory effect on the activation of HSCs.

Support or Funding Information

USDA Hatch CON00972
Twenty‐Four‐Hour Urine Phosphorus is Highly Variable in Patients with Moderate Chronic Kidney Disease on a Controlled Phosphorus Intake
Authors: Stremke Elizabeth R. , McCabe Linda D. , McCabe George P. , Martin Berdine R. , Wastney Meryl , Moe Sharon M. , Weaver Connie M. , Peacock Munro , Hill Gallant Kathleen M.
Published: November 2023

Twenty‐four‐hour urine phosphorus (24h uP) is commonly considered a good reflection of dietary phosphorus (P) intake in healthy adults in P balance because net absorption of P is linearly related to dietary intake over a wide range of intakes. This relationship has been assumed to also apply to patients with chronic kidney disease (CKD). However, factors other than dietary P intake and intestinal P absorption may affect 24h uP excretion, including the rate of net bone retention and soft tissue P deposition and retention. This study is a secondary analysis of the variation in 24‐hour urine phosphorus and its reliability as a measure of phosphorus intake in patients with moderate chronic kidney disease. Thirteen consecutive 24‐hour urines from 2‐week balance studies in 8 patients with estimated glomerular filtration rates 26–53 mL/min/1.73m2 on a tightly‐controlled dietary phosphorus intake of 1564 ± 52 mg/d were analyzed for variability and reliability. Results show, while consuming a controlled P diet, there is wide day‐to‐day intrasubject (standard deviations from 105–208 mg/d or %CV 15–60) and intersubject variation in 24h uP (standard deviation of 267 mg/d or %CV =37), as well as phosphorus/creatinine ratio. Intraclass correlation coefficients were calculated for 24h uP (r = 0.65) and phosphorus/creatinine ratio (r = 0.60). From these values, it was estimated that the number of replicates needed to have at least 75% reliability for 24h uP or P/Cr is 2 (95% CI: 1,5), and more replicates if a greater degree of reliability is desired. While 75% is generally considered a threshold for an acceptable level of reliability, the 13 replicates that were available from these balance studies gave over 90% reliability in the measures of both 24h uP and P/Cr. Dietary phosphorus intake calculated from 24‐hour urine phosphorus underestimated intake in some patients by up to 98% and overestimated intake in others by up to 79%. In patients with moderate chronic kidney disease, 24h uP is highly variable and its use can lead to erroneous conclusions regarding dietary phosphorus intake.

Support or Funding Information

The parent study was a randomized controlled trial funded by Sanofi/Genzyme through an unrestricted investigator‐initiated grant to MP, and, in part, by a NCRR Clinical and Translational Sciences Award (U54‐RR025761, Shekhar, PI). This secondary analysis was supported through institutional funds of KMHG. KMHG is supported by NIH K01 DK102864.
Mixture of Oleanolic acid and Ursolic Acid Mimicked the Effects of Fructus Ligustri Lucidi on Calcium Kinetics, Bone Properties, and Serum 1,25(OH)2D3 in Ovariectomized Rats
Authors: Wong Man Sau , Cao Si Si , Wastney Meryl E. , Lachcik Pamela J. , Weaver Connie
Published: November 2023

Oleanolic acid (OA) and ursolic acid (UA) are major chemical constituents in Fructus Ligustri Lucidi (FLL), a Chinese herb previously shown to improve calcium balance and bone properties in rats. OA and UA have been reported to stimulate osteoblastic differentiation and inhibit osteoclast formation in vitro. However, it is unclear if they are the bioactive ingredients that account for the in vivo actions of FLL on Ca balance and bone properties. The present study was designed to study if OA or OA+UA mimicked the effects of FLL on bone and calcium homeostasis using ovariectomized (OVX) rats. Three‐month‐old female Sprague Dawley rats were subjected to ovariectomy and after a two‐month stabilization period, were randomly assigned to 4 groups (n =13/group) fed with experimental diet containing FLL (700mg/kg/d), OA (25mg/kg/d), or OA (25mg/kg/d) + UA (8.3mg/kg/d) or control diet for 6 weeks. All diets contained 0.6% Ca, 0.65% P. Ca balance was measured in rats at week 1 and 6 and Ca kinetic study was performed at week 6 with 45Ca tracer. Calcium kinetics were modeled using WinSAAM software and a 3‐compartment model. At sacrifice, left femur and lumbar vertebra were collected for micro‐CT scanning and serum was obtained for 1,25(OH)2D3 measurement using a commercial kit. FLL, OA, and OA+UA diets significantly improved bone mineral density, bone volume to total volume, and trabecular thickness in femurs in OVX rats. Ca balance, however, was not altered in response to diet treatment at either week 1 or week 6 in OVX rats. As predicted by kinetic modeling, rats fed with OA and OA+UA diets deposited more calcium in bone and resorbed more calcium from the skeleton while rats fed with FLL and OA+UA diets decreased endogenous excretion of calcium as compared to those fed the control diet. All three treatments resulted in a higher mass of compartment 3 (vs. control group) due to changes of transfer rate between compartment 2 and 3, and appeared to be associated with their effects on increasing serum 1,25(OH)2D3 level in OVX rats. In summary, OA and OA+UA mimicked the effects of FLL on improving calcium utilization, protecting bone and modulating serum 1,25(OH)2D3 level in OVX rats, indicating that OA and UA are the bioactive ingredients that account for the in vivo actions of FLL.

Support or Funding Information

The study was supported by research fund (15100015) and research studentship for Miss Si Si Cao from the Hong Kong Polytechnic University as well as funding from National Science Foundation, PR China (81528024).
The effects of iron status on testosterone biosynthesis and testicular antioxidant capacity in male rats
Authors: Tsao Chih‐Wei , Liao Yuan‐Ru , Liew Yih‐Fong , Lin Ying‐Hung , Hsu Yu‐Juei , Liu Chin‐yu
Published: November 2023

Iron is an essential element for almost all living organisms as it participates in a wide variety of metabolic processes, including oxygen transport, deoxyribonucleic acid (DNA) synthesis, and electron transport. Previous study found that iron deficiency may lead to decreasing spermatozoa vitality, DNA damage, and oxidative stress. However, few studies did describe the relationships between the iron deficiency and testicular functions were not well‐known. The aim of this study is to explore the association between iron deficiency and hypogonadism. Thirty‐two weaning male Wistar rats were divided into iron‐adequate control group (AI), moderate iron‐deficient group (MID), severe iron‐deficient group (ID) and severe iron‐deficient pair‐fed group (IPF). The rats are fed the diet with different content of iron for four weeks and then sacrificed after fasting for 12 hours. Iron deficiency caused a significant reduction in serum testosterone, 3β‐hydroxysteroid dehydrogenase (3β‐HSD) and CYP11A1 protein. The expression of Bax/Bcl2 and c‐PARP were induced in the ID group. The hematoxylin‐eosin staining of ID group that the diminished seminiferous tubule sizes and degenerated germinal cells was observed. In addition, SOD (superoxide dismutase) level in testis tissues was also decreased in the ID group. Iron deficiency may lead to reduction of testosterone biosynthesis due to induction the oxidative stress and apoptosis in the testis tissue.

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MOST 104‐2314‐B‐016‐029, Ministry of Science and Technology, Taiwan
Loss of keratins mimics p38MAPK‐mediated pathogenic effects of autoantibodies in pemphigus
Authors: Vielmuth Franziska , Walter Elias , Loschke Fanny , Magin Thomas M. , Spindler Volker , Waschke Jens
Published: November 2023

Desmosomes are adhesive complexes required for strong intercellular adhesion. Desmosomal cadherins including desmoglein (Dsg) 1 and 3 serve as adhesion molecules while plaque proteins maintain the anchorage to the intermediate filament cytoskeleton, the latter of which is composed of keratins in the epidermis. The collapse and detachment of the keratin filament network from desmosomes are hallmarks of the bullous autoimmune dermatosis pemphigus vulgaris (PV), which is caused by autoantibodies against Dsg1 and Dsg3. However, the significance of these keratin filament alterations for loss of cell cohesion in response to PV autoantibody (PV‐IgG) binding is largely unknown. To investigate the relevance of keratins for pemphigus pathogenesis we used murine keratinocytes lacking all keratin filaments (KtyII k.o.) and compared them with wildtype (KtyII wt) controls. Keratin‐deficient keratinocytes displayed reduced intercellular adhesion and Dsg3‐mediated binding events were weaker compared to KtyII wt as demonstrated by dissociation assays and atomic force microscopy (AFM) adhesion measurements, respectively. AFM and FRAP experiments revealed higher mobility of Dsg3 molecules of KtyII k.o. cells. Interestingly, although depletion of Dsg3 in response to PV‐IgG occurred faster, incubation with pathogenic autoantibodies had only a minor additional effect on cell adhesion in keratin‐deficient cells compared to wt. This may be explained by robust p38MAPK activation in KtyII k.o. cells in the resting state, which is a central signaling pathway in pemphigus. Nevertheless, anisomycin‐mediated p38MAPK activation further reduced cell cohesion showing that loss of keratins alone does not completely abolish keratinocyte adhesion. Inhibition of p38MAPK reinforced intercellular adhesion in keratin‐deficient keratinocytes and increased binding strength of Dsg3 interactions under basal conditions and blocked PV‐IgG‐induced loss of cell cohesion. These results indicate that keratins regulate p38MAPK signaling which in turn reduces Dsg3 binding properties. Moreover, loss of keratins mimics effects of autoantibodies indicating that keratin reorganization is essential for impaired cell cohesion in pemphigus.
Intestinal Mucosa Pro‐Repair Properties of Macrophage Derived IL‐10 are Mediated by CREB Triggered Epithelial WISP‐1 Signaling
Authors: Quiros Miguel , Nishio Hikaru , Neumann Philipp A , Leoni Giovanna , Garcia‐Hernandez Vicky , Siuda Dorothee , Feng Mingli , Bernal Gabriela , Hilgarth Roland , Williams Holly , Brazil Jennifer C , Dedhia Priya H , Spence Jason , Parkos Charles A , Denning Timothy L , Nusrat Asma
Published: November 2023

In response to injury, epithelial cells migrate and proliferate to cover denuded mucosal surfaces and repair the barrier defect. This process is orchestrated by a dynamic cross‐talk between immune cells and the epithelium. However, the underlying mechanisms of repair remain incompletely understood. Here, we report that increased generation of IL‐10 in healing wounds orchestrates repair of the intestinal mucosal barrier. Conditional deletion of IL‐10 specifically in CD11c‐expressing cells in‐vivo identified macrophages as a critical innate immune contributor to IL‐10‐mediated mucosal wound repair. Consistent with these findings, wound closure in Rag1 KO mice was unimpaired, suggesting that adaptive immune cells do not influence intestinal mucosal wound repair. Furthermore, following mucosal injury, macrophage‐derived IL‐10 resulted in epithelial cAMP response element‐binding protein (CREB) activation and subsequent synthesis and secretion of the pro‐repair WNT1‐inducible signaling protein 1 (WISP‐1). WISP‐1 promoted epithelial proliferation and wound closure by activation of epithelial beta catenin and cyclin D1. These findings identify an important role of macrophages derived IL‐10 that cross‐talks with epithelial WISP‐1 signaling to orchestrate mucosal repair.

Support or Funding Information

This work was supported by NIH grants (RO1DK055679, RO1DK089763 to A. Nusrat; R01DK097256 to T. Denning; and DK61739, DK72564 and DK79392 to C.A. Parkos); the Crohn's and Colitis Foundation of America Research Fellowship Award (326912) to M. Quiros
Desmoglein 2 Regulates the Intestinal Epithelial Barrier via p38 Mitogen‐Activated Protein Kinase
Authors: Ungewiß Hanna , Vielmuth Franziska , Suzuki Shintaro T. , Maiser Anderas , Harz Hartmann , Leonhardt Heinrich , Kugelmann Daniela , Schlegel Nicolas , Waschke Jens
Published: November 2023

Intestinal epithelial barrier properties are maintained by a junctional complex consisting of tight junctions (TJ), adherens junctions (AJ) and desmosomes. Desmoglein 2 (Dsg2), an adhesion molecule of desmosomes, is required for epithelial barrier properties and may contribute to barrier defects in Crohn's disease. Here, for the first time we identified extra desmosomal Dsg2 on the surface of differentiated enterocytes displaying junctional complexes by Triton extraction, confocal microscopy, SIM and STED. Atomic force microscopy (AFM) revealed Dsg2‐specific binding events on the surface of enterocytes with a mean unbinding force of around 30 pN which were more prominent close to cell borders. Furthermore, we demonstrated that inhibition of Dsg2 binding induced activation of p38 mitogen‐activated protein kinase (p38MAPK) which is a key regulator of desmosomal adhesion. An inhibitory Dsg2 antibody activated p38MAPK, under conditions where it significantly reduced cell cohesion in a dissociation assay and Dsg2‐specific binding in AFM. Loss of Dsg2 in enterocytes led to reduced p38MAPK activity accompanied with loss of barrier integrity and impaired barrier reformation. Dsc2 rescue in Dsg2/Dsc2 deficient enterocytes did not restore p‐p38MAPK levels indicating, that Dsg2 regulates the activity of this kinase. Direct activation of p38MAPK in Dsg2 knockout cells enhanced barrier reformation demonstrating that Dsg2‐mediated activation of p38MAPK is crucial for barrier function. Collectively, our data show that Dsg2, in addition to its adhesion function, regulates intestinal barrier function via p38MAPK signaling.

Support or Funding Information

This work was supported by the DFG priority program SPP 1782
Epigenetic re‐programming of breast cancer by pharmacological targeting of C‐terminal binding protein
Authors: Byun Jung S , Gardner Kevin L
Published: November 2023

The C‐terminal binding protein (CtBP) is a family of dimeric nuclear proteins whose levels are increased in cancers of the colon, ovaries, prostate and breast. Elevated CtBP protein expression is associated with poor cancer survival, thus implicating CtBP as both a biomarker and a promising candidate for therapeutic intervention. As a dimer, CtBP provides a scaffold that couples multiple different DNA‐binding transcriptional regulators with a variety of chromatin modifying protein complexes to alter the epigenetic landscape throughout the nucleus. These properties provide the rationale for pharmacological targeting of CtBP to alter or epigenetically change regulated gene expression in cancer cells. In this study, we employ computer assisted drug design to screen for optimal quantitative structure‐activity relationships (QSARs) between small molecules and CtBP to identify 24 potential CtBP inhibitors. Functional screening of these compounds identifies 4 lead compounds with low toxicity and high water solubility. Treatment of breast cancer cells at micro‐molar concentrations of these small molecular inhibitors induces significant de‐repression of epigenetically silenced pro‐epithelial genes in the mesenchymal, triple negative breast cancer cell line, MDA‐MB‐231. This re‐activation is associated with eviction of CtBP from the respective gene promoters, disrupted recruitment of CtBP‐chromatin modifying protein complexes, increased deposition of activating epigenetic histone marks, and upregulation of both pro‐epithelial gene mRNA and protein expression. In functional assays, CtBP inhibition by these small molecular inhibitors decreases cellular invasion, and improves DNA repair.

FRET (Förster resonance energy transfer) analysis demonstrates that CTBP inhibition results in decreased FRET intensity, suggesting the CTBP dimerization interaction is repressed by CTBP inhibition. In addition, pharmacological inhibition of CtBP combines with established epigenetically targeted drugs to synergistically decrease cell migration and potentiate the reactivation of silenced pro‐epithelial gene expression in triple negative cancer cells. Finally, CTBP inhibition results in transcriptional repression of MDR1 expression and reduces the population of Doxorubicin resistant cells in triple negative breast cancer cell lines.

These findings implicate the possible use of this compound in strategies for therapeutic intervention that may increase the efficacy and decrease the acquired resistance to targeted therapeutic intervention in breast cancer.
DNA Methylation, Hydroxymethylation and Formylation in Human Frontal Cortex of Autistic and Schizophrenic Subjects
Authors: Trivedi Malav , Deth Richard , Zhang Yiting , Abdolmaleky Hamid
Published: November 2023

Epigenetic regulation provides a molecular mechanism for long‐lasting, activity‐dependent changes in gene expression. In the brain, increased neuronal activity can translate into changes in DNA and histone methylation with epigenetic consequences, contributing to neuroplasticity and memory formation. The folate and vitamin B12‐dependent enzyme methionine synthase (MS) controls methylation and is sensitive to redox status, linking aerobic metabolism to epigenetic regulation. In previous postmortem brain studies we showed that MS expression in frontal cortex decreases dramatically with age (500‐fold), while levels of the MS cofactor methylB12 (methylcobalamin; MeCbl) decrease more than 10‐fold across the lifespan. Furthermore, in autistic subjects MS expression and MeCbl levels were significantly lower than age‐matched controls, in association with decreased levels of the methyl donor S‐adenosylmethionine (SAM). MeCbl levels were also decreased in schizophrenia subjects. To determine whether these abnormal metabolic conditions are associated with epigenetic manifestations, we measured global levels of 5‐methylcystosine (5mCyt), 5‐hydroxymethylcytosine (5hmCyt) and 5‐formylcytosine (5fCyt) in DNA from Brodmann areas 10/11 and 44/45 in frontal cortex. Levels of 5mCyt and 5hmCyt were higher in autistic subjects (4–11 yrs) vs. age‐matched subjects (5–13 yrs), while levels of 5fCyt were lower. In schizophrenic subjects (36–49 yrs), levels of 5mCyt were lower than age‐matched controls (36–50 yrs), while levels of 5hmCyt and 5fCyt were higher. A comparison of young vs. middle‐age control subjects revealed significantly lower levels of 5mCyt and 5fCyt, while levels of 5hmCyt were higher in the older subjects. Methylation of specific CpG sites in the D4 dopamine receptor (DRD4) gene were significantly elevated in autism and schizophrenia. These findings confirm that abnormally decreased MS expression and MeCbl levels in frontal cortex of autistic and schizophrenic subjects are associated with differential 5mCyt, 5hmCyt and 5fCyt status, suggesting that epigenetic dysregulation may contribute to neurodevelopmental and neuropsychiatric disorders.

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None