bluuebunny/crossref_metadata_2025 huggingface dataset:
Consuming A Whey Protein Supplement Two Hours After A Low Protein Breakfast Preserves Amino Acid Availability
Authors: Hudson Joshua L. , Paddon‐Jones Douglas , Campbell Wayne W.
Published: November 2023

Thirty grams of high quality dietary protein is generally needed to maximally stimulate muscle protein synthesis. However, this generally exceeds the amount of protein consumed at breakfast, in part, due to practical limitations. This issue may be mitigated by consuming an easily preparable whey protein supplement. Amino acids from a whey protein supplement appear sooner in circulation than amino acids from mixed meals. As a result, consuming a whey protein supplement after a low protein‐containing meal may be an effective and convenient means to preserve available amino acids. Therefore, the primary aim of this proof‐of‐concept study is to assess whether consuming a 20 g whey protein supplement 120 min after consuming a 10 g protein breakfast would result in peak and composite postprandial amino acid responses that were not different to a 30 g protein mixed meal breakfast alone. In a randomized, crossover design, 12 subjects (6 males, 6 females; mean ± SEM; age: 29 ± 1 y; BMI: 26.0 ± 1.0 kg/m2) completed three 5‐h acute postprandial feeding trials in which they consumed mixed meal breakfasts containing 10 g of dietary protein (LP), 30 g of dietary protein (HP), and 10 g of dietary protein with an additional 20 g of whey protein isolate consumed 120 minutes after the mixed meal breakfast (WP). Blood samples were collected at times −30, 0, 30, 60, 120, 150, 180, 240, and 300 min relative to breakfast intake. Total amino acid (TAA) and essential amino acid (EAA) concentrations peaked 180 min after breakfast consumption in both the WP and HP trials, with greater peak concentrations in the WP trial than in the HP trial. Branched chain amino acid (BCAA) and leucine concentrations peaked earlier in the WP trial than in the HP trial (180 versus 240 min, respectively) and were greater in the WP versus HP trials. Composite iAUCpos over 300 min for TAA and all the amino acid subfractions were greater in the HP than LP trial, during which the postprandial amino acid concentrations did not increase above baseline. HP and WP trial composite iAUCpos over 300 min were not different for TAA, EAA, and BCAA and leucine iAUCpos was ~91% greater in the WP than HP trial. Therefore, the WP trial iAUCpos may be predominantly attributable to the whey protein supplement consumed 2 hours after the low‐protein containing breakfast. In conclusion, there was no increase in amino acid availability when consuming a 10 g protein breakfast and amino acid availability was not different when either consuming a single isonitrogenous high protein‐containing breakfast or supplementing whey protein 2 hours after a low protein‐containing breakfast. Consuming a whey protein supplement 2 hours after a low‐protein breakfast may provide a convenient method to maximally stimulate muscle protein synthesis without having to consume a high protein‐containing breakfast.

Support or Funding Information

Purdue University, Ingestive Behavioral Research Center and Lynn Fellowship to JLH
Post‐prandial protein handling following ingestion of different amounts of protein during post‐exercise recovery in older males
Authors: Holwerda Andrew M. , Paulussen Kevin J. M. , Overkamp Maarten , Goessens Joy P. B. , Kramer Irene‐Fleur , Wodzig Will K. W. H. , Verdijk Lex B. , van Loon Luc J. C.
Published: November 2023

Purpose

The age‐related decline in skeletal muscle mass is, at least partly, attributed to anabolic resistance to food intake. Resistance‐type exercise sensitizes skeletal muscle tissue to the anabolic properties of amino acids. Data are warranted to define the amount of ingested protein needed to maximize post‐exercise myofibrillar protein synthesis rates in older individuals.

Methods

In a parallel group design, forty‐eight healthy older men (66±1 y) were randomly assigned to ingest 0, 15, 30 or 45 g milk protein concentrate (MPC80) after performing a single bout of resistance type exercise. Post‐prandial protein digestion and absorption kinetics, whole body protein metabolism and myofibrillar protein synthesis rates were assessed using primed, continuous infusions of L‐[ring‐2H5]‐phenylalanine and L‐[ring‐2H2]‐tyrosine combined with the ingestion of intrinsically L‐[1‐13C]‐phenylalanine labeled milk protein.

Results

A total of 76±2% (11.4±0.3 g), 63±3% (18.9±0.9 g) and 60±3% (26.8±1.2 g) of the protein derived amino acids were released in the circulation during 6 h after ingesting 15, 30 or 45 g protein (P<0.01). Ingestion of 15, 30 and 45 g protein resulted in higher whole‐body protein synthesis when compared to the control treatment (0.61±0.01, 0.64±0.01 and 0.67±0.02 vs 0.53±0.02 μmol·Phe·kg−1·min−1, respectively; P<0.01). Whole body protein breakdown rates were lower after ingestion of 45 g when compared with 0 g (0.45±0.01 vs 0.52±0.02 μmol·Phe·kg−1·min−1; P<0.01). Whole‐body protein oxidation rates were increased after ingestion of 45 g when compared with 15 g and 0 g protein (0.061±0.003 vs 0.050±0.003 and 0.046±0.002 μmol Phe·kg−1·min−1, respectively; P<0.05). Whole‐body protein balance increased in a dose‐dependent manner after the ingestion of 0, 15, 30 and 45 g protein (0.02±0.0, 0.11±0.0, 0.16±0.01, and 0.22±0.01 μmol·Phe·kg−1·min−1, respectively; P<0.001). Muscle tissue analyses are currently being performed.

Conclusions

Dietary protein ingested after resistance‐type exercise is rapidly digested and absorbed, with 60–75% of the protein derived amino acids being released into the circulation within 6 h after ingestion. Whole body protein synthesis rates and net protein balance are increased in a dose‐dependent manner following the ingestion of 15, 30 and 45 g milk protein in older males.
Evaluation of an Innovative Feeding Toolkit to Improve Complementary Feeding and Child Growth in Rural Malawi
Authors: Faerber Emily C. , Ko Jasmine , Weiss Jennifer , Girard Amy Webb
Published: November 2023

Background

Inadequate complementary feeding (CF) is recognized as a cause of growth failure, but there is considerable variability in the ability of interventions to impact growth through CF practices.

Objective

To evaluate the effectiveness of a feeding toolkit which aims to improve child growth through improved meal frequency, meal volume and food consistency in rural Malawi.

Methods

Households in Mchinji district, Malawi with a child aged 6 to 17 months were selected via single stage cluster sampling based on caregivers' participation in community women's groups; 923 households from 53 communities completed a baseline survey in July/August 2015 and an endline survey in June 2016. In October/November of 2015, women's groups were randomized to either the intervention or control, with households in intervention communities eligible to receive a feeding toolkit consisting of a feeding bowl with demarcations and symbols that cued age‐appropriate meal frequency and volume; a slotted spoon designed to promote optimal porridge consistency; and nutrition education. Caregivers in the control groups received nutrition education. Logistic and linear regression was used to assess the impact of the feeding toolkit on CF practices and changes in child weight‐ and height‐for‐age (WAZ, HAZ) and weight‐for‐height (WHZ) z‐scores adjusted for covariates.

Results

The mean child age at endline was 21.3 ± 3.3 months, with 51% female. Receipt of the toolkit was associated with increased meal volume (β=30 ml, 95% CI 14 ml, 46 ml) but not with the odds of meeting minimum dietary diversity (P=0.6) or minimum acceptable diet (P=0.7). There was evidence of interaction between toolkit receipt and food security for minimum meal frequency (P=0.06) and consistency (P=0.07). The toolkit improved odds of minimum meal frequency (OR=1.55, 95% CI 1.10, 2.16) and was associated with increased meal thickness (β=0.24, 95% CI 0.12, 0.36) but only among moderate or severely food insecure households. There was a trend toward improved HAZ in children who received the toolkit (β=0.12, 95% CI=−0.01, 0.26, P = 0.07), but there were no associations with WAZ (P=0.6) or WHZ (P=0.3).

Conclusion

A feeding bowl and spoon with nutrition education was associated with improved meal volume in all households, and improved meal frequency and consistency in the most food insecure households. Further studies are warranted to understand the impact of such an intervention on linear growth.

Support or Funding Information

Funding and support for this research was provided by Concern Worldwide; Malawi Department of Nutrition, HIV and AIDS; World Bank; Emory Global Health Institute; Laney Graduate School, Emory University; and the National Institute of Diabetes and Digestive and Kidney Disease of the National Institutes of Health under award number T32DK007734.
Polyphenolic Compounds Downregulate IL‐6 in Gut Epithelial Cells under Inflammatory Conditions
Authors: Washburn Karley F. , Crockett Erica K. , Graef Jennifer L. , Lucas Edralin A. , Smith Brenda J.
Published: November 2023

The majority of immune cells in the human body reside within gut‐associated lymphoid tissues. Gut epithelial cells serve as an important barrier between foreign substances in the lumen of the intestine and these immune cell populations. Polyphenolic compounds found in plant‐based foods are well known for their immune modulating properties; however, the bioavailability of these compounds can be limited, leaving their mode of action in question. This study examined the alterations in cytokine production in gut epithelial cells exposed to polyphenolic compounds extracted from a fruit rich in polyphenols (i.e., dried plums), under normal and inflammatory conditions. The MODE‐K epithelial cell line, which was derived from C3H/HeJ mice, was used in these studies. Cells were cultured in RPMI GlutMAX media, supplemented with 10% FBS, 1% sodium pyruvate, 1% non‐essential amino acids, 1% HEPES, and 0.1% β‐mercaptoethanol and incubated in 5% CO2/95% air‐humidified at 37° C. The polyphenol extract (DP‐PP) was prepared from dried plum powder (‘Improved French’) in 80% ethanol, followed by filtration and further purification using a Waters C‐18 column. The resulting extract was lyophilized, weighed, and re‐suspended in dimethyl‐sulfoxide (DMSO) and filtered for cell culture use. The dose‐dependent effects of DP‐PP (0, 2.5, 5, 10, 20, 40, & 80 μg/mL) and tumor necrosis factor‐alpha (TNF‐α; 0, 0.1, 1.0, & 10 ng/mL) on cell proliferation and viability were assessed over time using the methyl‐thiazolyldiphenyl‐tetrazolium (MTT) and the trypan blue exclusion assays. No significant decreases in cell growth and viability were observed with DP‐PP (0 to 40 ug/mL) and TNF‐α (0 to 1.0 ng/mL). For gene expression analyses, MODE‐K cells were plated at 1×106 cells/well. After 24 hrs the cells were treated with TNF‐α (0 or 1ng/mL) for 30‐min, followed by treatment with DP‐PP (0, 5, 10, or 20ug/mL). RNA was extracted at 2 and 24 hrs, and RT‐PCR was performed to assess gene expression of interleukin 6 (Il6) and transforming growth factor‐beta (tgfb) under normal and inflammatory conditions. The data were analyzed utilizing a factorial approach with TNF‐α and DP‐PP as factors. Main effects were observed with Il6 expression only, in that TNF‐α up‐regulated Il6 and DP‐PP attenuated this response. Under normal conditions, DP‐PP had no significant effect on Il6, but DP‐PP dose‐dependently suppressed (p<0.05) the TNF‐α‐induced increase in Il6 expression at 2 hrs. By 24 hrs, this response had normalized. DP‐PP did not affect the expression of Tgfb under normal or inflammatory conditions. Our findings indicate that DP‐PP can counteract a pro‐inflammatory state in the gut mucosa by down regulating IL‐6, which can have implications for intestinal health.
Intestinal Microbiota and Host Metabolism Respond Differentially in Lean and Obese Individuals Following Six‐Week Consumption of Galloyl Derivatives from Mango (Mangifera Indica L.) Pulp
Authors: Kim Hyemee , Barnes Ryan , Fang Chuo , Talcott Stephen , Mertens‐Talcott Susanne
Published: November 2023

Obesity‐associated inflammation has been an increasing concern in public health. This study investigated changes in the gut microbiome, pharmacokinetics/pharmacodynamics of galloyl metabolites, and anti‐inflammatory activities in lean and obese subjects in a randomized pilot study. Twelve lean (Average BMI=22.34) and nine obese (Average BMI=33.42) subjects were enrolled and administrated 400g of mango daily for six weeks. Plasma samples were collected at 0, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 hours after mango consumption on days 1 and 42. Gut microbiota composition in stool samples was measured using qPCR and biomarkers for inflammatory conditions in plasma were determined using a multiplex bead assay. After six weeks of mango consumption, plasma concentrations of galloyl metabolites increased in all subjects. Plasma in lean subjects contained 2.4 times higher levels of polyphenols compared to obese subjects, including pyrogallol derivatives. As shown in qPCR, the levels of pyrogallol‐producing microbiota, Aspergillus oryzae and Lactococcus lactis were significantly lower in obese subjects compared to lean subjects on the baseline, but the levels were increased in obese subjects to match the lean subjects after six weeks. The levels of Bifidobacterium Spp. were not significantly changed, but directly correlated with the amount of galloyl metabolites found in plasma (p=0.0243, r=0.36, Spearman correlation). The levels of Bacteroides thetaiotaomicron and Clostridium leptum, microbiota associated with obesity, were significantly higher in obese subjects compared to lean subjects on the baseline, but decreased after six weeks of mango intake. In obese subjects, plasma concentrations (AUC0‐8h) of IL‐8 and MCP‐1 were significantly decreased after six weeks, while there were no changes in lean subjects. The plasma concentrations (AUC0‐8h) of IL‐10 were significantly higher in lean subjects compared to obese subjects and correlated with the amount of galloyl metabolites found in plasma (p=0.0218, r=0.38, Spearman correlation). These results indicate that obese individuals do not exhibit the same level of adaptive absorption and metabolism as lean subjects, but still benefit from a decrease of pro‐inflammatory cytokines (IL‐8 and MCP‐1). The observed changes may in part be due to differences in gut microbiota composition and metabolism.

Support or Funding Information

Partial funding for this research was provided by: National Mango Board
A prospective study of taste and smell dysfunctions and the risk of hypertension
Authors: Liu Yi‐Hsuan , Huang Zhe , Vaidya Anand , Li Junjuan , Chen Yichi , Curhan Gary , Wu Shouling , Gao Xiang
Published: November 2023

Background

Previous studies have shown that olfactory receptors, which mediate smell chemosensation, are also located in the kidney and involved in regulating blood pressure. Mammalian epithelial sodium channel located in taste receptor cells has also been found to participate in blood pressure regulation. However, there is currently no human study that has examined the association between taste and smell functions and hypertension development.

Objective

We prospectively examined the association between taste and smell functions and the risk of hypertension in a community‐based cohort. We hypothesized that participants with taste and smell dysfunctions had higher risk of developing hypertension.

Methods

The study included 6,482 Chinese adults (5,114 men and 1,368 women) who were free of hypertension in 2012 (baseline). Chronic taste and smell dysfunction data were collected in 2012 by using questionnaire containing self‐reported problems with the sense of taste and smell from National Health Interview Survey. Newly developed hypertension was evaluated in 2014. Odds ratios (ORs) and 95% confidence intervals (CIs) of hypertension, across three categories of dysfunction, based on the number of taste and smell dysfunctions (ranged from 0 to 2), were calculated by using logistic regression, after adjusting for potential confounders, including age, sex, education level, income status, work environment, physical activity level, salt intake, smoking status, alcohol consumption, body mass index (BMI), waist circumference, diabetes, dyslipidemia, C‐reactive protein (CRP), glomerular filtration rate (GFR), and blood pressure in 2012.

Results

We documented 1,338 incident hypertension cases within two‐year period. After adjusting for potential confounders, individuals with both taste and smell dysfunctions had increased risk of developing hypertension (adjusted OR=2.25; 95% CI: 1.08–4.69) and faster increase in systolic blood pressure (p<0.05) relative to those without taste and smell dysfunction.

Conclusion

We found that participants with self‐reported taste and smell dysfunctions had higher risk of developing hypertension independent of known risk factors, suggesting an association between chemosensory functions and blood pressure regulation.
Association between dietary inflammatory potential and risk of breast cancer: findings from the Sister Study
Authors: PARK YONG‐MOON , Shivappa Nitin , Petimar Joshua , Steck Susan , Hébert James , Sandler Dale
Published: November 2023

Background

It has recently been suggested that the inflammatory potential of diet may influence the risk of breast cancer. However, few studies have investigated associations for pre‐versus post‐menopausal breast cancer or considered effect modification by obesity.

Methods

Energy adjusted‐Dietary Inflammatory Index (E‐DII)TM was calculated for 49,731 Sister Study cohort participants who completed a validated 146‐item food frequency questionnaire at enrollment in 2003–2009. Women aged 35 to 74 years in the U.S. and Puerto Rico were eligible if they had a sister who had been diagnosed with breast cancer. High DII scores represent a more pro‐inflammatory diet. Cox proportional hazards models were used to estimate multivariable‐adjusted hazard ratios (HR) and 95% confidence intervals (CIs) for breast cancer risk by E‐DII quartiles, after adjusting for potential confounders including known risk factors for breast cancer. Breast cancer subtypes were based on estrogen receptor (ER) and progesterone receptor (PR) status. Time‐varying menopausal status was considered.

Results

During follow‐up (mean, 7.6 years), 2,155 breast cancers were diagnosed at least 1 year after enrollment. E‐DII scores were not associated with overall or invasive breast cancer (HRhighest vs. lowest quartile: 1.11 [95% CI, 0.96–1.29]; 1.14 [95% CI, 0.95–1.35], respectively), although there was a positive association between E‐DII scores and ER‐PR‐breast cancer (HRhighest vs. lowest quartile: 1.69 [95% CI, 1.02–2.80], Ptrend = 0.06). E‐DII scores were associated with increased risk of postmenopausal breast cancer (HRhighest vs. lowest quartile: 1.20 [95% CI, 1.01–1.42], Ptrend = 0.04) and ER‐PR‐invasive postmenopausal breast cancer (HRhighest vs. lowest quartile: 2.06 [95% CI, 1.19–3.58], Ptrend = 0.01). E‐DII scores were not associated with breast cancer in premenopausal women. Obese women (i.e., those with a body mass index ≥30 kg/m2) had increased risk of overall and invasive breast cancer with increasing E‐DII scores (HRhighest vs. lowest quartile: 1.42 [95% CI, 1.07–1.90], Ptrend = 0.04; 1.54 [95% CI, 1.11–2.13], Ptrend = 0.02, respectively), which was not observed in non‐obese women. Similarly, E‐DII was more strongly associated with breast cancer among women with waist circumference ≥ 88 cm.

Conclusion

Our findings suggest that dietary inflammatory potential may differentially influence the risk of breast cancer by menopausal status, breast cancer subtype, and adiposity phenotypes.

Support or Funding Information

This research was supported by the Intramural Research Program of the National Institutes of Health, the National Institute of Environmental Health Sciences (Z01‐ES044005).
High dietary acid load is associated with risk of breast cancer: findings from the Sister Study
Authors: Park Yong‐Moon , Steck Susan , Fung Teresa , Merchant Anwar , Sandler Dale
Published: November 2023

Background

Recently, diet‐induced acid–base imbalance was shown to be associated with metabolic disorders such as insulin resistance and type 2 diabetes. It also has been suggested that diet‐induced acidosis might promote carcinogenesis or tumor progression through influencing molecular activity such as insulin growth factor. However, there is little or no epidemiologic evidence to support a role of diet induced acidosis in cancer risk.

Methods

We used data from 49,731 Sister Study participants who completed a validated 146‐item food frequency questionnaire at enrollment in 2003–2009. Women aged 35 to 74 years in the U.S. and Puerto Rico were eligible if they had a sister who had been diagnosed with breast cancer. The potential renal acid load (PRAL) considering body surface area was used to estimate diet‐induced acid load, based on daily intake of protein, phosphorus, potassium, calcium, and magnesium calculated from the food frequency questionnaire. Cox proportional hazards models were used to estimate multivariable adjusted hazard ratios (HR) and 95% confidence intervals (CI) for breast cancer risk by PRAL quartiles, after adjusting for potential confounders including known risk factors for breast cancer.

Results

We identified 2,155 breast cancers at least 1 year after enrollment during follow‐up (mean, 7.6 years). The highest PRAL quartile, reflecting greater acid‐forming potential, was associated with increased risk of overall and invasive breast cancer, compared with the first quartile (HR: 1.21 [95% CI, 1.06–1.39], Ptrend = 0.21; 1.18 [95% CI, 1.00–1.38], respectively). When stratified by time‐varying menopausal status, this association persisted only in postmenopausal breast cancer (HRhighest vs. lowest quartile: 1.29 [95% CI, 1.11–1.50], Ptrend = 0.02; 1.22 [95% CI, 1.03–1.45], respectively). The association tended to be more pronounced in estrogen receptor (ER) negative, progesterone receptor (PR) negative invasive breast cancer (HRhighest vs. lowest quartile: 1.68 [95% CI, 1.06–2.67], Ptrend = 0.08), compared with ER+PR+ invasive breast cancer (HRhighest vs. lowest quartile: 1.14 [95% CI, 0.93–1.40], Ptrend = 0.91), although there was no statistically significant interaction effect. These associations tended to be weaker, but consistent, using the net endogenous acid production (NEAP) score, an alternative index of diet‐induced acid load based on protein and potassium intake. No differential association was observed for normal or overweight/obese body mass index.

Conclusion

Our findings suggest that diet‐induced acidosis may be a risk factor for breast cancer, especially in postmenopausal breast cancer. Our results need to be confirmed in other populations and further research is warranted to understand the underlying mechanisms.

Support or Funding Information

This research was supported by the Intramural Research Program of the NIH, the National Institute of Environmental Health Sciences (Z01‐ES044005).
Predictors of Carotenoid Concentrations in Human Serum: Role of the Intestinal Microbiome
Authors: Djuric Zora , Bassis Christine , Ren Jianwei , Chan Rena , Kato Ikuko , Ruffin Mack T. , Sen Ananda , Turgeon D. Kim
Published: November 2023

Carotenoids from fruits and vegetables are suggested to reduce the risks of several cancers. Inter‐individual variability in serum carotenoid concentrations is large, and is only modestly explained by dietary intakes. There is evidence that the colonic microbiota can maximize the bio‐accessibility of carotenoids by digestion of plant cell walls. Here we evaluated if differences in the relative abundance of bacterial taxa in the colon could contribute to the inter‐individual variability in serum concentrations of carotenoids. We utilized data and samples from a dietary intervention study that enrolled healthy individuals at increased risk of colon cancer. Colon mucosal biopsies were obtained without prior preparation of the bowels, and 93% of the biopsies were successfully amplified for the V4 region of the 16s rRNA gene. The relative abundance of bacterial phyla in the colonic mucosa of 89 individuals was: 6.7% Proteobacteria, 60% Firmicutes, 27% Bacteroidetes, 1.9% Actinobacteria and 2.8%Verrucomicrobia. At baseline, higher serum concentrations and higher dietary intakes of carotenoids were both associated with a lower abundance of Firmicutes taxa, unlike the direct association of higher body mass index (BMI) with increased abundance of Firmicutes. Higher serum carotenoids also were positively associated with measures of bacterial diversity in contrast to the negative association of diversity with BMI. Surprisingly, there was little change in bacterial taxa post‐intervention with either a Mediterranean diet or a Healthy Eating diet. Subsequent analyses evaluated the contributions of the microbiome, diet and demographic factors to the inter‐individual variation in serum carotenoid concentrations at baseline. Correlations of individual serum carotenoids with bacterial Operational Taxonomic Units indicated that oxygenated carotenoids, namely xanthophylls, segregated from other carotenoids. In linear regression models with serum xanthophyll concentrations as the outcome, bacterial taxa accounted for an additional 11% of the variance after controlling for BMI, smoking, and dietary xanthophyll intakes, and all these factors together accounted for 35% of the inter‐individual variability in serum xanthophylls. For serum beta‐carotene, bacterial taxa contributed an additional 4% of the variance after controlling for BMI, smoking, and dietary intakes. Regression models for alpha‐carotene and lycopene were not improved by including bacterial abundance. These results are consistent with a significant role for colonic microbiota in accessibility of specific carotenoids from fruits and vegetables in humans, and especially for xanthophylls which are high in fruits and green, leafy vegetables.

Support or Funding Information

We acknowledge support from NIH grants RO1 CA120381, T32 CA009676, P30 CA046592, P60 DK20572, P30 DK089503, UL1RR024986, and the Rose and Lawrence C. Page, Sr. Family Charitable Foundation.
Effect of Mixed‐Carotenoid Supplementation and Physical Activity on Irisin and Adiponectin: A 6‐Month Double Blind Placebo‐Controlled Pilot Study in Children with Obesity
Authors: Canas Jose A. , Lochrie Amanda , McGowan Amy Galena , Schettino Christopher , Hossain Jobayer , Balagopal Babu
Published: November 2023

Body

Short term supplementation with mixed‐carotenoids (MCA) and physical activity reduces the accrual of abdominal subcutaneous white adipose tissue (scWAT) in children with obesity. Presumptive mechanisms may involve the induction of thermogenic beigeing and myo‐adipocytokines such as irisin, high‐molecular weight adiponectin (HMW‐ADI) and leptin.

Method and Design

Of the 20 obese children recruited (Age = 126 ± 5 months), 17 completed a randomized double blind placebo controlled study involving one hour of physical activity for 10‐days of intense life‐style intervention followed by 6 months of usual lifestyle. Patients were randomized to MCA (Jarrow Formulas CaroteneAll®)5000 IU of beta‐carotene (BC) and alpha carotene; 20 mg of lutein; 4 mg of zeaxanthin, 20 mg of lycopene; 1000 mcg of astaxanthin; and 20 mg of gamma tocopherol vs. placebo at entry and were sampled at baseline, three weeks and 6 months. BC measured by HPLC, leptin measured by RIA and HMW‐adiponectin and irisin were measured by ELISA. The percent change in visceral (vWAT) and scWAT was determined by quantitative cross‐sectional multi‐slice MRI (1.5‐T; GE Medical Systems) at baseline and 6 months.

Results

BC was inversely related to BMI Z‐score, vWAT and scWAT (p<0.05 for all). Supplementation resulted in 95 ± 66% increase in BC at 6 months as compared to 3 ± 19% decrease in the placebo group (p=0.001). Pair‐wise comparisons for treatment effects based on estimated marginal mean ± SEM showed there was a 4.1±1.7 ng/mL (p=0.033) increase in irisin at three weeks and a 4.8±1.8 ng/ml (p=0.018) increase in HMW‐adiponectin at six month between supplement and placebo groups after Bonferroni adjustments for age, gender and baseline BMI z‐score. There was a 4% decrease in mean scWAT by MRI in the supplement group (p=0.016) after six months.

Conclusion

MCA significantly increased irisin after 10 days of physical activity and HMW‐adiponectin after 6 months of supplementation as compared to placebo. These changes may contribute to the decrease in scWAT accrual at 6 months mediated by the induction of thermogenic beigeing signals. Further studies are warranted.

Support or Funding Information

The Players Center for Child Health at Wolfson Children's Hospital
Salmonella typhimurium‐derived Virulence Protein Overcomes Multidrug Resistance in Tumors
Authors: Mercado‐Lubo Regino , Hang Gang , McCormick Beth A.
Published: November 2023

We have recently developed a novel therapeutic platform for the treatment of cancers that over‐express the multidrug resistance transporter P‐glycoprotein (P‐gp), a major impediment for the success of conventional chemotherapy. This technology is based on our discovery that a Salmonella typhimurium‐derived virulence protein, SipA, not only functionally down‐regulates P‐gp, but also potentiates the cytotoxic effect of chemotherapeutics such as doxorubicin and vinblastine in vitro by significantly increasing the intracellular accumulation of the drugs. To safely deliver SipA in our in vivo models of cancer, we engineered a carboxyl modified 15nm gold nanoparticle (AUNP) packed with a SipA corona to create a SipA‐AuNP biomimetic. We found that SipA‐AuNPs, when used in combination with doxorubicin, promote tumor regression in a subcutaneous model of murine colon cancer, as well as a patient‐derived xenograft mouse model of breast cancer. We are currently working to identify the specific SipA domain responsible for the down‐regulation of P‐gp. Thus far, SipA has been truncated into two fragments: a SipAb C‐terminal fragment426–684 (259 AA), which binds actin, and a SipAa fragment2–425 (424 AA), known for its ability to induce pro‐inflammatory responses. When used in in vitro models of colon cancer, the SipAa fragment exhibits a conserved ability to down‐regulate P‐pg (70% reduction compared to the negative control), whereas the SipAb fragment fails to reduce P‐gp. Concomitantly, we are evaluating the ability of SipA to increase the efficacy of other known P‐gp substrates, such as etoposide, daunorubicin and paclitaxel, since this effect is specific to drugs efluxed by P‐gp. Using a cell proliferation assay, in which human colon carcinoma cells are cultured in media containing etoposide with or without purified‐SipA, we have found that those with SipA exhibit a profound reduction in the IC50 (50.20±0.30 to 8.05±1.15 μM). Given that we observe improved cytotoxicity of drugs effluxed by P‐gp, we are now implementing novel patient‐derived xenograft mouse models of cancers that use P‐gp substrates (i.e. doxorubicin and etoposide) as the first line of treatment, such as of B‐cell lymphomas and ovarian cancer. We believe that SipA could present a significant advancement as an adjuvant therapy when compared to previous P‐gp reversal agents. Furthermore, SipA can potentially facilitate repurposing old cancer drugs and help reduce the amount the chemotherapeutics needed for effective treatment, thereby reducing the side effects associated with chemotherapy.

Support or Funding Information

National Institutes of Health Grants DK56754 and DK33506 awarded to Beth A. McCormickThe Worcester Foundation, and by a UMCCTS Pilot Project Program award (CTSA Grant # UL1TR000161) awarded to Beth A. McCormick Massachusetts Medical School start‐up fund and National Institutes of Health grant R01MH103133
Role of PMNs in inhibition of DNA repair and induction of genomic instability
Authors: Butin‐Israeli Veronika , Mehl Lindsey , Mascarenhas Lorraine Lorraine , Sumagin Ronen
Published: November 2023

PMNs play key roles in host defense and tissue homeostasis, however, en‐masse tissue infiltration by PMNs often leads to aberrant inflammation and tissue injury, and is a hallmark of inflammatory bowel diseases (IBD). High numbers of mucosa infiltrating PMNs have been correlated with the severity of IBD, whereas IBD patients were found to be at significantly higher risk of developing colorectal cancer (CRC). While the link between inflammation and cancer is well established, little is known about PMN contribution to carcinogenesis. The accumulation of DNA damage is one of the leading causes of carcinogenesis. Activated PMNs can induce DNA damage via generation of reactive oxygen species (ROS). However, in the current work we describe a novel mechanism whereby the release of membrane‐derived microparticles (MPs) by tissue infiltrating/activated PMNs resultes in potent, ROS‐independent accumulation of double stranded DNA breaks (DSBs). PMN‐MP treatment of intestinal epithelial cells (IECs) induced persistent activation of DNA damage responses, including post‐translational modification of protein 53BP1, activation of DNA‐dependent protein kinase (DNA‐PKcs) and phosphorylation of histone H2AX, indicating increased generation of DSBs. Moreover, PMN‐MP treatment (96 hours) resulted in cell cycle arrest in S‐phase, deficient elongation of replication fork and persistent accumulation of γH2AX/53BP1 nuclear foci, suggesting inhibition of Homologous Recombination (HR)‐dependent DSB repair. Importantly, pre‐treatment of PMN‐MPs with RNAse significantly attenuated PMN‐MP‐mediated inhibition of DSB repair suggesting a role for micro‐RNAs (miRNAs) in this process. Indeed, a screen for potential miRNAs candidates with a known role in the regulation of DNA damage/repair responses revealed a significant upregulation of miR‐155 in PMNs following activation with IFNγ or TNFα, and its release in PMN‐MPs. MiR‐155 has been previously suggested to target a key regulator of HR, RAD51, which was found to be downregulated following PMN‐MP treatment. Importantly, an addition of miR‐155 antagonists reversed the loss of RAD51, partially recovered HR and decreased accumulation of DSBs induced by PMN‐MPs. Finally, in an in vitro model simulating PMN induced inflammation (continued co‐culture of IECs with PMN‐MPs over 12 population doublings) we detected an increase in aneuploidy and a high frequency of chromosomal aberrations indicating induction of genomic instability. Thus, our studies show for the first time that PMNs, via secretion of miRNA‐rich MPs, promotes genomic instability through the inhibition of HR, which may lead to mutagenesis and inactivation of tumor suppressor genes, and cellular transformation.

Support or Funding Information

This work was supported by grants from the National Institutes of Health (NIH) DK101675 as well as American Cancer Society IRG‐9303718
Altered Expression of Neuropeptide Receptors in the Bleomycin (Bleo)‐Induced Mouse Model of Scleroderma (SSc)
Authors: Parra James , Ibrahim Mohamed , McKinnon Elizabeth , Levinson Howard , Sunday Mary E
Published: November 2023

Study Objective

Background

SSc is a chronic collagen‐vascular disease that initially presents with dermal fibrosis, later progressing to multiple organ fibrosis. There is no treatment to arrest SSc. Recently, a mouse model of SSc was reported, in which Bleo is injected intradermally (ID) for 21–28 days. Two groups showed that reactive oxygen species (ROS) cause this dermal fibrosis. We previously observed that ROS trigger GRP‐mediated pulmonary fibrosis induced by hyperoxia or radiation. We first verified that ROS trigger this dermal fibrosis, and now test the hypothesis that gastrin‐releasing peptide (GRP) from cutaneous nerves has a role in this process by acting on myofibroblasts [alpha‐smooth muscle actin, SMA+] and pericytes [SMA+ and neural/glial antigen 2, NG2+]. We tested expression of the two GRP receptors, GRPR and neuromedin B receptor (NMBR), by using immunohistochemistry.

Methods

10‐wk old C3H/HeJ females were injected ID on the flank with Bleo (100‐μg) 5d/wk for 3‐wks. Some Bleo‐injected mice also received antioxidant N‐acetylcysteine (NAC) IP, or GRP blocking mAb‐2A11. After 21d, sections of skin lesions were immunostained for SMA, NG2, GRPR, or NMBR. Relative extent of immunostaining in dermis and epidermis was scored on a scale from 0–3, comparing prevalence of (+) cells (0, 1=detected in few cells, 2=many cells (+), & 3=most cells positive. ImageJ analysis (immunopositive area as a percentage of tissue volume) was used to confirm scoring results.

Results

Pericytes & myofibroblasts were significantly increased in areas of dermal thickening, with NG2 and SMA immunostaining linearly correlated (R2 = .87. P<0.05). Dermal SMA & NG2 were reduced by NAC (~80% decrease, P<0.001) or mAb2A11 (~50% decrease, P<0.01), similar to measurements of dermal thickness. Epidermal GRPR was decreased by over 50% in Bleo+2A11 mice compared to Bleo alone (P < 0.005). There was no other difference in GRPR between study groups. NMBR expression was generally unchanged between the groups.

Conclusion

In the Bleo mouse model of SSc, increased pericytes and myofibroblasts occur in dermal fibrosis. Although either GRPR &/or NMBR could contribute to Bleo‐induced dermal fibrosis their expression in dermis is unchanged between groups. Sustained epidermal expression of both receptors would be consistent with potential GRP signaling in epidermis as a mechanism leading to epidermal hyperplasia and dermal fibrosis, possibly via epithelial‐mesenchymal transformation.

Support or Funding Information

This work was supported by a P30 NIH Pilot Grant Award to M. Sunday by the Duke Skin Disease Research Center [Dr. Russell Hall, Program Director].
Assessing Gliogenesis in a Murine Multifactorial Brain Injury Model System
Authors: Domowicz Miriam S. , Wadlington Natasha L. , Henry Judith G. , Diaz Kasandra , Munoz Miranda J. , Schwartz Nancy B.
Published: November 2023

Brain injury in humans caused by trauma, intrauterine infections, hypoxia and/or ischemia, pre‐maturity or low birth weight, during the perinatal period which coincides with glial cell (astrocyte and oligodendrocyte) maturation in the brain, often has devastating neurological consequences such as epilepsy, cerebral palsy, and behavioral and cognitive problems. Due to the variety of risk factors associated with these neurodevelopmental disorders, we are using a multifactorial injury model to examine the effects on differentiation and maturation of the two glial subtypes. The model consists of penetrating perinatal traumatic brain injury, with or without intraperitoneal injection of lipopolysaccharide (LPS) as a model of remote pathogen exposure. Postnatal‐day‐6 (P6) mouse pups underwent sham, LPS‐alone, stab‐alone, or stab‐plus‐LPS treatments. At different ages after injury, the pups were harvested and gene‐expression changes for astrocyte, neuron, oligodendrocyte, and precursor‐cell markers were assessed using mRNA in situ hybridization (ISH) and qPCR. In general, the injured (ipsilateral) side exhibited enlarged ventricles, occasional disruption of the cortex structure and sporadic cysts; whereas the uninjured (contralateral) side remained unaffected overall, comparable to sham‐treated animals. Astrocyte‐precursor and mature‐astrocyte marker mRNA levels increased in the stab‐alone and stab‐plus‐LPS treated animals. Microglial/macrophage markers CD208 and CD68 levels, increased in the ipsilateral sides of stab and stab‐plus LPS animals by P10, but the differences resolved by P15. Decreased mRNA expression of mature oligodendrocyte markers, proteolipid protein 1 (PLP) and myelin basic protein (MBP) and the neuronal marker Class III beta‐tubulin (TUBB3), was observed only on the ipsilateral side in stab‐treated pups by ISH. Interestingly, ectopic expression of the neural stem cell marker SRY(sex‐determining region Y)‐box 2 (SOX2), astrocyte precursor SOX9, and the oligodendrocyte precursor marker oligodendrocyte transcription factor (OLIG2) mRNAs were observed in the ipsilateral side following stab injuries with or without LPS treatment at P10. By fluorescent ISH, partial colocalization of SOX2, OLIG2 and SOX9 mRNAs with GFAP‐positive cells was confirmed. These data suggest that shortly following traumatic injury, concomitant with the decrease in mature oligodendrocyte and neuronal marker expression, neural stem cells and glial precursor populations may be attempting to repopulate the injury region.

Support or Funding Information

Supported by U.S. Public Health Service Grants HD009402 and GM066522
How the cytosol‐to‐membrane translocation kinetics and signaling of PKCγ are dysregulated in the neurodegenerative spinocerebellar ataxia type14 (SCA14)
Authors: Aslam Naveed , Alvi Farah
Published: November 2023

The spinocerebellar ataxia type 14 (SCA14) is an autosomal neurodegeneration disease that is clinically characterized by the progressive ataxia of gait accompanied by slurred speech and abnormal eye movements. These symptoms are linked to the loss of Purkinje cells leading to cerebellar neurodegeneration. The onset age of this disease is in the range of 33–43 years. The MRI results clearly show the atrophy of cerebellum and autopsy results indicates the reduction in number of Purkinje cells as well the size and complexity of their dendritic arbor. Observations in Purkinje cells link PKCγ to the SCA14 disease and its gene PRKCG is now widely recognized as a disease‐causing gene. PKCγ. The PKCγ is a brain specific molecule and is highly expressed in cPCs. Observations also show that PKCγ link to SCA14 is established more through gain‐of‐function and in fact both positive and negative regulation of PKCγ expression and activity could result in the changes in number, size and complexity of dendritic arbors in Purkinje cells. Here, we investigate the question that why the membrane residence time of PKCγ is decreased in SCA14 compared to wildtype (WT) cPCs. We explore this question by proposing two signaling models for PKCγ; First model describes the signaling mechanism of how could the activity of WT PKCγ be regulated in cPCs, whereas the second model shows the signaling of mutant PKCγ in SCA14 associated cPCs. Both these models show that in response to extracellular stimuli‐induced depolarization at membrane, both PKCγ and DGKγ translocate to the membrane. Our results also indicate that for same set of parameters the membrane residence time of PKCγ is shorter compared to WT molecule. These results explain that membrane residence time of PKCγ is regulated by DAG and the translocated molecule returns to cytosol as the DAG levels decrease. These results suggest that even though all the kinetic parameters are same in WT & mutant models the residence time of PKCγ is reduced in mutant model due to reduced signaling. This study shows that for the same strength of extracellular signal the membrane life time of mutant PKCγ is decreased due to constitutively active mutant PKCγ molecule in cytosol. This cytosolic molecule in turn, leads to the phosphorylation and activation of DGKγ molecule inside cytosol. Thus, mutant model explains that when both the DAG effector molecules are found in their active states in cytosol their interactions in membrane compartment are reduced thus, reducing the residence time of PKCγ molecule in membrane compartment in mutant model.

Support or Funding Information

Privately Funded
Deficiency of AMP‐Activated Protein Kinase Alpha 1 Promotes the Vulnerability of Atherosclerotic Plaques at Brachiocephalic Arteries
Authors: DING YE , Zhang Miao , Song Ping , Zou Ming‐hui
Published: November 2023

Background

Atherosclerotic plaque rupture is the most important mechanism responsible for the sudden and unpredictable onset of acute coronary syndromes. We had previously demonstrated that genetic deletion of AMP‐activated protein kinase (AMPK), a master sensor and regulator of energy and redox homeostasis, instigates endothelial dysfunction and accelerates atherosclerosis in mice in vivo. In addition, we have reported that genetically inactivate AMPKα2 in Vascular Smooth Muscle Cells (VSMC) aggravates atherosclerotic plaque instability. However, the roles of AMPKα1, another essential isoform of AMPKα, with respect to atherosclerotic plaque stability remains unclear.

Objective

The aim of this study was to evaluate the specific impact of AMPKα1 on atherosclerotic plaque stability in the brachiocephalic arteries (advanced atherosclerosis), and to elucidate the underlying mechanisms.

Methods and Results

Atherosclerotic plaque stability were monitored in the brachiocephalic arteries isolated from Apoe−/− or Apoe−/−AMPKα1−/− mice fed western diet for 10 weeks. We identified that Apoe−/− AMPKα1−/− mice showed multiple features indicative of unstable atherosclerotic plaque relative to Apoe−/− mice. The plaques located in the brachiocephalic artery of Apoe−/−AMPKα1−/− mice displayed higher incidence of intra‐plaque hemorrhage, the presence of buried fibrous caps, and discontinuity of the fibrous caps compared to those found in Apoe−/− mice. Further, the necrotic core sizes and macrophage contents were significantly increased in Apoe−/−AMPKα1−/− mice relative to those from Apoe−/− mice. Conversely, VSMC contents, fibrous cap thickness and plaque collagen contents, which were widely used as indicators of stable plaque, were markedly decreased in Apoe−/−AMPKα1−/− mice when compared to those in Apoe−/− mice. Importantly, the production and activity of macrophage derived MMP9, which is a critical factor in the rupture of unstable atherosclerotic plaques, were significantly increased in brachiocephalic arteries and aortas from Apoe−/−AMPKα1−/− mice. This result was further confirmed in Bone Marrow Derived Macrophages (BMDM) isolated from AMPKα1 deletion mice. Furthermore, we found that AMPKα1 deficiency promoted VSMC apoptosis both in the plaque and in cultured cells. We identified that AMPKα1 deletion increases expression of pNF‐κB p65 both in BMDM and in VSMCs, suggesting that AMPKα1 deletion activates NF‐κB pathway. NF‐κB inhibition with pharmacological inhibitor or siRNA‐mediated silencing of NF‐κBp65 abolished the increase of MMP9 in macrophage and VSMC apoptosis induced by AMPKα1 deficiency.

Conclusions

Taken together, this study demonstrates that AMPKα1 deficiency result in increased activity of macrophage MMP9 and induces VSMC apoptosis, which subsequently promotes atherosclerotic plaque instability in NF‐κB‐dependent manner.
Production of TLRs triggered pro‐inflammatory cytokines through calcium dependent and independent pathways in HaCaT cells
Authors: Kim Eunok , Ha Injin , Kim Kyuseok , Park Dain , Lee Min Young
Published: November 2023

Keratinocytes can be activated by Propionibacterium acnes (P. acne) and lead to the production of pro‐inflammatory cytokines. P. acnes stimulates the production of pro‐inflammatory cytokines via Toll‐like receptor2 (TLR2) and TLR4. In this study, we examined the effects of P. acne on production of cytokines via TLR2 and TLR4 in the HaCaT cells. First, we compared the calcium release from HaCaT, immortalized keratinocytes and THP‐1 monocytes after stimulation with TLR ligands. Our results show that during inflammatory reaction, TLR2 activation with TLR2 ligand increase intracellular calcium influx but not TLR4 in HaCaT. However activation with both of TLR2 and TLR4 ligands increase intracellular calcium influx in THP‐1 monocytes cells. These data suggest that only TLR2 activation in HaCaT cells induce calcium increase different from THP‐1. Furthermore RT‐PCR analysis revealed that TLR2 stimulation induces expression of the epidermal differentiation marker, Keratin 1. These results demonstrate that TLR2 stimulation by P. acnes induce abnormal keratinization and that TLR2 and TLR4 activation increase production of pro‐inflammatory cytokines (IL‐1a, IL‐1b, IL‐6 and IL‐8) in HaCaT cells. The production of pro‐inflammatory cytokines and chemokines in keratinocytes can recruits macrophage cells into keratinocytes and induces inflammation. In conclusion, these results suggest that production of pro‐inflammatory cytokines by TLR2 and TLR4 in HaCaT cells is induced through TLR2 induced calcium dependent and TLR4 induced calcium independent pathway.

Support or Funding Information

This study was supported by a grant from the Traditional Korean Medicine R&D Project, Ministry of Health & Welfare, Republic of Korea. (HI16C0801)