bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Three Doses of Dietary Sialyllactose on Growth and Intestinal Development in the Neonatal Piglet
Authors: Monaco Marcia Helena , Wang Mei , Pan Xiao , Li Qian , Richards James D. , Chichlowski Maciej , Berg Brian , Dilger Ryan N , Donovan Sharon M.
Published: November 2023

Oligosaccharides are the third most abundant component in human milk and modulate neonate microbiota, gut and immune development. This study investigated potential dose‐dependent effects of sialyllactose (Lacprodan SAL‐10®, SL) on growth and gut development of neonatal piglets. Beginning at 2 d of age, 38 vaginally‐derived male piglets (mean body weight (BW) 1.64 ± 0.04 kg) were randomized to diets formulated to contain: control (CONT) [0 mg SL/L milk replacer], low (LOW) [130 mg SL/L], moderate (MOD) [380 mg SL/L], and high (HIGH) [760 mg SL/L]. Diets contained 4 g/L of a 1:1 mixture of polydextrose and galactooligosaccharides. Diets were prepared at 20% (wt/vol) and piglets received 285 mL and 325 mL per kg BW on d2–5 and 5–33, respectively. On d32 or 33, piglets were euthanized and samples were collected. Outcomes were analyzed using a one‐way ANOVA; significance was set at p ≤ 0.05 and trends at 0.05 < p < 0.10. Dietary SL was well tolerated regardless of dose and growth patterns were similar among the groups. Weight gain between d2 and 33 did not differ among the groups and the average gain was 4.23 ± 0.29 kg (CONT: 4.19 kg ± 0.42; LOW: 4.08 kg ± 0.64; MOD: 4.43 kg ± 0.69; HIGH: 4.22 kg ± 0.68). Similarly, intestinal length and weight, villus and crypt morphology in the small and large intestine and the number of acidic sulfated mucin‐secreting goblet cells did not differ among the groups. Short‐chain and branched‐chain fatty acid profiles in colonic contents were similar between all four groups. No differences in complete blood counts were observed and serum clinical chemistry values were similar (minerals, electrolytes, protein, enzymes, kidney function, liver function and acid:base balance and prothrombin time), except for activated partial thromboplastin time (aPTT) and glutathione dehydrogenase (GLDH). APTT (seconds) in HIGH (12.6 ± 0.5) was lower (p<0.02) than CONT (14.1 ± 0.2), LOW (13.8 ± 0.4) and MOD (13.6 ± 0.2). GLDH (U/L) in CONT (0.9 ± 0.1) was lower (p<0.02) than LOW (1.3 ± 0.1) and MOD (1.5 ± 0.2) and tended (p<0.09) to be lower than HIGH (1.2 ± 0.08). Due to the low magnitude of differences and lack of a clear dose response, the differences in aPTT were not clinically significant. In addition to a lack of dose‐response in our study, normal values for other liver enzymes suggest that slightly higher GLDH values are also unlikely of clinical significance. In conclusion, dietary SL at up to 760 mg/L was well tolerated and supported similar growth and intestinal development as the CONT formula in neonatal piglets.

Support or Funding Information

Mead Johnson Nutrition
Using Metatranscriptomics to Determine Effects of Dietary Supplementation with Bovine Milk Oligosaccharides in Healthy Adults
Authors: Westreich Samuel Taler , Barile Daniela , Salcedo Jaime , Mills David A , Smilowitz Jennifer T. , Korf Ian , Lemay Danielle G
Published: November 2023

The study of human milk has revealed the presence of complex human milk oligosaccharide (HMO) molecules, believed to help encourage formation and development of the infant's gut microbiome. HMOs function as prebiotics for beneficial bacteria, block attachment to pathogens, and interact directly with intestinal cells. Many bovine milk oligosaccharides (BMOs) are structurally analogous to HMOs and can be obtained from whey streams currently discarded as waste. In a prior study, we fed BMOs to healthy adults for the first time to determine safety and tolerability, and found that they did not cause more bloating or gas than a placebo, which suggests that BMOs may be fermented more slowly than other prebiotics. Using 16S sequencing, we also found that fecal microbial populations did not change significantly.

It is possible for the functional activity of a microbial population to change without overall abundances changing. We therefore investigated the activity of all gut microbes in vivo simultaneously in a subset of these humans (n=6) fed BMOs. RNA was extracted from RNA‐preserved stool and libraries were prepared and sequenced to yield a minimum of 40 million reads per subject. Intra‐individual bacterial transcriptional profiles appeared more similar to each other. However, differential abundance analysis revealed a small, but significant (p < 3.23e‐41), increase in overall Bacteroides transcriptional activity in response to the BMO supplement. Specific functions of Bacteroides species are being further investigated. In conclusion, our results demonstrate that it is possible for gut microbe populations to remain stable while their functional activities change in response to diet, emphasizing the need for a metatranscriptome‐based approach in future studies.

Support or Funding Information

Funding was provided by Arla Food Ingredients.
Maternal BMI is correlated with non‐fasting human milk insulin, but not macronutrients
Authors: Sarangam Maithri L , Young Bridget , Garces Ana Lucia , Figueroa Lester , Tejada Gabriela , Kemp Jennifer , Westcott Jamie Lucia , Hambidge Michael , Krebs Nancy
Published: November 2023

Background

The impact of maternal phenotype on bioactive components of human milk (HM) remains unclear. This is particularly relevant given the increasing worldwide prevalence of maternal obesity and insulin resistance. In particular, the relationships between maternal BMI, macronutrients, and HM insulin concentration requires further study.

Methods

We analyzed 126 HM samples collected at 14 days postpartum from participants in the “Women First” study in Guatemala, which administered small quantity lipid‐based nutrient supplements from either the preconception or prenatal period until delivery. The RCT is ongoing and treatment arms are unknown. All maternal supplementation concluded at birth. HM samples were collected via hand expression, after the infant initiated let‐down. Skim milk was generated via centrifugation and HM insulin was analyzed via chemiluminescence immunoassay. Macronutrients and total calories were analyzed via human milk analyzer (Miris) in 102 milk samples.

Results

In the cohort as a whole, HM lactose = 6.9 ± 0.6 g/100mL; protein = 1.2 ± 0.4 g/100mL; fat = 3.9 ± 1.3 g/100mL; calories = 70.4 ± 12.0 kcal/100mL; insulin 14.3 ± 9.0 μIU/mL. Neither maternal BMI nor time‐of‐day (between 09:00 and 16:30) were related to any of the macronutrients or HM caloric density. Mean HM insulin concentration was significantly different across maternal pre‐pregnancy BMI categories (p<0.002). Mean HM insulin concentrations were 12.4 ± 6.6 μIU/mL, 15.3 ± 10.0 μIU/mL, and 21.5 ± 13.0 μIU/mL, for the normal (n=71), overweight (n=40), and obese (n=14) BMI categories respectively. Neither HM macronutrient concentrations nor caloric density were related to HM insulin concentrations. In multivariable regression, both maternal pre‐pregnancy BMI and the time‐of‐day of HM collection were significantly and positively related to HM insulin values (p<0.001). Together these variables explained 16% of the variation in HM insulin.

Conclusions

Our findings suggest that maternal pre‐pregnancy BMI likely impacts the concentration of insulin in HM. This finding is particularly relevant given that prandial state was not controlled in our collection methods. The impact of higher concentrations of HM insulin on the recipient infant requires further investigation.

Support or Funding Information

Bill & Melinda Gates Foundation OPP1055867; University of Colorado Clinical and Translational Research Centers NIH/NCATS UL1‐TR001082; NIH: NICHD and Office of Dietary Supplements #U10 HD076474
Maternal Factors and Human Milk Oligosaccharide Composition in the CHILD Cohort
Authors: Robertson Bianca Marie , Bode Lars , Sharma Atul K. , Becker Allan B. , Mandhane Piushkumar J. , Subbarao Padmaja , Turvey Stuart E. , Lefebvre Diana L. , Sears Malcolm R. , Azad Meghan B.
Published: November 2023

Rationale

Accumulating evidence suggests that human milk oligosaccharides (HMOs) impact infant health. However, which maternal factors influence HMO composition is largely unknown.

Methods

We analyzed 427 breast milk samples from the Canadian Healthy Infant Longitudinal Development (CHILD) study by rapid high‐throughput HPLC to determine associations between HMO composition and maternal age, ethnicity, parity, secretor blood group status, method of delivery and milk collection time postpartum. Milk samples were collected at 4 months postpartum (median 16 weeks, interquartile range 14–19 weeks). Spearman correlations, t‐tests and multivariable linear regression were used to evaluate relationships between HMO composition and maternal factors.

Results

Maternal secretor blood group status, lactation time postpartum, and parity were significantly and independently associated with total HMO concentration. Secretor mothers, expressing an active FUT2 gene, had increased levels of total HMOs when compared to non‐secretors (mean 15,908 ± 1,513 vs. 8.935 ± 2,796 nmol/mL, p<0.001). Non‐secretor status was more common among Asian vs. Caucasian mothers (40% vs. 26%, p=0.04). Total HMO level decreased with increasing time postpartum (adjusted b: −66 nmol/L per week; 95%CI −113, −19; p=0.006), although some individual HMOs increased, including: 3′‐fucosyllactose (3′FL, Spearman r = +0.15), 3′‐sialyllactose (3′SL, +0.18), lacto‐N‐fucopentaose‐III (LNFPIII, +0.12), and especially disialyllacto‐N‐tetraose (DSLNT, +0.28) (all p<0.02). Furthermore, total HMO levels increased with parity (adjusted b: +529 nmol/mL per child; 95%CI 173, 884; p<0.01). In contrast, maternal age and method of delivery were not significantly associated with total HMO level.

Conclusion

Our data suggest that both genetic and non‐genetic maternal factors influence HMO composition. Whether these associations have implications for infant health remains to be investigated.

Support or Funding Information

This study was funded by Research Manitoba and supported by the Canadian Institutes of Health Research and the Allergy, Genes and Environment Network of Centres of Excellence.
Investigating Methods of Estimating the Rate of Synthesis of Breast Milk and Milk Fat
Authors: Geddes Donna Tracey , Gardner Hazel , Lai Ching‐Tat , Hartmann Peter Edwin , Kent Jacqueline Coral
Published: November 2023

Study objective

Currently measurement of the average rate of breast milk synthesis and fat synthesis relies on a 24‐h milk profile (test‐weighing all breastfeeds and expressions over a 24‐h period and collecting small milk samples before and after each breastfeed for the same period). It has been demonstrated that when the breasts are expressed using an electric breast pump each hour, the volume of the fourth expression is equivalent to the hourly rate of milk synthesis. We aimed to confirm this finding, and ascertain if the same technique could be used to estimate the rate of milk fat synthesis.

Methods

The study was approved by the Human Research Ethics Committee of The University of Western Australia and all participants provided written informed consent. Mothers (n = 15) measured their 24‐h milk profile, and then expressed their breast(s) using a Medela Symphony breast pump for 10 min on arrival at the research room, and 1, 2 and 3 hours later. The volume of milk expressed each time was measured and small (<1 mL) milk samples collected before and after each expression. The creamatocrit of each sample was measured and converted to fat using the formula fat (g.L−1) = 3.968 + (5.917*creamatocrit). Linear mixed models were used to compare the volumes and fat content at each hourly expression with the hourly milk yield and fat synthesis from the 24‐h milk profile. Random effect was the mother.

Results

The mean rates of milk synthesis were 17.8 mL.h−1 (sem 2.0) and 19.0 mL.h−1 (sem 2.0) for 24‐h milk profile and fourth expression, respectively, and there was a significant relationship between the two measurements (P=0.002). The slope of the regression was 0.66 with a confidence interval of 0.32 – 1.00). The mean rates of fat synthesis were 0.75 g.h−1 (sem 0.06) and 0.96 g.h−1 (sem 0.13) for 24‐h milk profile and fourth expression, respectively, and there was a significant relationship between the two measurements (P=0.039). The slope of the regression was 1.14 with a confidence interval of 0.20 – 2.08.

Conclusion

These data confirm that the fourth hourly expression can be used to estimate the average rate of breast milk synthesis for individuals. However, while there is an overall relationship between the two methods for estimating the rate of synthesis of milk fat, the wide confidence interval for the slope of the regression implies that this technique is less useful for individuals.

Support or Funding Information

Unrestricted research grant from Medela AG
Impact of Maternal Diet and Exercise on Human Milk Insulin
Authors: Burchenal Clare , Barbour Lynda , Hernandez Teri , Friedman Jed , Krebs Nancy , Young Bridget
Published: November 2023

The impact of maternal phenotype, diet and exercise on bioactive components in human milk (HM) remains an area of active research. We aimed to determine if maternal diet, specifically the nature of her carbohydrate intake, correlated with the fasting concentrations of insulin in her breast milk. Maternal 3‐day diet records were collected at 2‐weeks and 4‐months postpartum from 48 exclusively breastfeeding women. Diets were analyzed by a registered dietitian at the Colorado Clinical and Translational Sciences Institute using the Nutrient Data System for Research (NDSR) software (2012). Fasting maternal blood and a mid‐feed HM sample were collected in the morning hours at 2‐weeks and 4‐months. Skim milk was generated via centrifugation and HM and maternal plasma insulin concentrations were measured via chemiluminescent ELISA. At 4‐months, mothers completed a Godin Leisure‐Time Exercise Questionnaire to characterize degree of physical activity. The cohort exhibited a wide range of caloric and fiber intakes. Maternal circulating insulin concentrations were highly correlated with HM insulin at both time points (p<0.0001). Increased consumption of insoluble fiber at 2‐weeks was associated with decreased concentrations of HM insulin (p=0.049, n=48, R2=0.08). While this association did not remain significant at 4‐months, increased consumption of added sugars and %kcal from carbohydrates at 4‐months were both positively associated with HM insulin concentrations (n=37; p=0.075, R2=0.07 and p=0.044, R2=0.08, respectively). These relationships between dietary carbohydrate quality and fasting HM insulin remained significant after controlling for maternal pre‐pregnancy BMI, age, total calories and maternal insulin concentrations. Maternal exercise score was not related to HM insulin.

The quality of carbohydrate consumed at 2‐weeks and 4‐months postpartum was associated with HM insulin concentrations, suggesting that modifiable factors, such as diet, may impact bioactive components in HM. Recommendations to encourage fiber intake and limit added sugars may impact HM components; potential benefits to maternal health and infant outcomes warrant further investigation.

Support or Funding Information

University of Colorado Center for Women's Health Research; The Thrasher Research Fund; NIH/NIDDK: T32‐DK007658–21; NIH/NICHD F32‐HD0978068; The Colorado Clinical and Translational Sciences Institute NIH/NCATS UL1‐TR001082.
Quantity and Species of Fish Consumed Shapes Breast Milk Fatty Acid Concentrations Around Lake Victoria, Kenya
Authors: Fiorella Kathryn J , Milner Erin M , Omollo Dan O , Hickey Matthew D , Salmen Charles , Bukusi Elizabeth , Fernald Lia CH
Published: November 2023

Long‐chain polyunsaturated fatty acids found in breast milk are critical for optimal infant development and derived from dietary sources. While the availability of these fatty acids is associated with the consumption of fish in general, little is known about the relative contribution of maternal consumption of different fish species in shaping breast milk concentrations. Yet the availability of fish and composition of fish species are shifting in response to global environmental changes. Understanding how specific fish species contribute to breast milk fatty acid contribution provides an important link in understanding how the food environment affects nutrition.

We examined associations between fish consumption and fatty acid concentrations in breast milk among mothers living around Lake Victoria. We used cross‐sectional analyses of associations between recent fish consumption and breast milk fatty acid concentrations. The study was conducted around Lake Victoria on Mfangano Island, Kenya where multiple fish species are key dietary components and also widely exported. Breastfeeding mothers (n=60) contributed breast milk samples, anthropometric measurements, and questionnaire responses.

In the previous three days, 97% of women consumed an average of 178g +/− 111.1g of fish (~2 servings/3 days). Average breast milk concentrations of fatty acid were high, including docosahexaenoic acid (DHA; 0.75% of total fatty acids), eicosapentaenoic acid (EPA; 0.16%), arachidonic acid (AA; 0.44%), and alpha‐linoleic acid (0.54%). Breast milk DHA concentrations exceeded the global average of 0.32% in 98% of samples. We found that native fish, cichlids (Cichlidae) and dagaa (R. argentea), contribute high levels of DHA, EPA, and AA to local diets. We also found evidence for associations between fish species consumed and breast milk fatty acid levels of alpha‐linoleic acid (ALA), linoleic acid (LA), EPA, and AA when controlling for intake of other fish species, maternal BMI, maternal age, breastfeeding child age, and exclusive breastfeeding.

Our results provide evidence that the fish species consumed influences the fatty acid composition of breast milk. Ensuring access to diverse fish, and particularly inexpensive and non‐export species, may be important for diet quality as well as infant growth and development.

Support or Funding Information

NSF‐GEO grant CNH115057; Cornell University Atkinson Center for a Sustainable Future and NSF DDRI (KJF)
Effects of consuming different protein preloads on appetite, satiety, and subsequent food intake
Authors: Douglas Steve M , Gwin Jess A. , Leidy Heather J.
Published: November 2023

Background

Consistent data from acute, single preload studies demonstrate improvements in perceived appetite and satiety following the consumption of high‐protein preloads compared to normal protein preloads. However, it is currently unclear as to whether protein quality (i.e., protein source) influences these outcomes.

Objective

To examine whether the consumption of protein preloads (egg white isolate, whey isolate, micellar casein, and instant egg white) differentially modulate postprandial hunger, fullness, desire to eat, prospective food consumption, eating initiation, and subsequent food intake.

Methods

Twelve young adults (age: 22±1 y; BMI: 22.1±0.9 kg/m2) reported to the research facility between 7 – 9 am to complete each 4‐h testing day. A set of questionnaires assessing perceived hunger, fullness, desire to eat, prospective food consumption were completed followed by the consumption of the 130 kcal beverage preloads, varying in protein source. A water preload was also included as a control. The same set of questionnaires along with the assessment of whether they would like to eat again were completed every 30 min throughout the 4‐h postprandial period. After the 4‐h, the participants were given an ad libitum pasta meal to consume until ‘comfortably full.’

Results

The consumption of the protein preloads improved hunger, fullness, desire to eat, prospective food consumption and delayed the request to eat again vs. the control (water); however, no differences in appetite and satiety outcomes were observed between protein sources. No differences in energy content at the lunch meal were observed between any preloads.

Conclusions

When comparing high‐quality animal‐based proteins consumed as a beverage preload, relatively no differences in markers of appetite control and satiety were detected, suggesting that protein quality has little impact on these outcomes when consuming 30g of protein.

Support or Funding Information

Rembrandt Foods
Post‐Exercise Consumption of Whole Eggs or Egg Whites Improves Whole Body Leucine Balance but Does Not Differentially Modulate Leucine Kinetics in Resistance‐Trained Young Men
Authors: Martinez Isabel G. , van Vliet Stephan , Shy Evan L. , Beals Joseph W. , Ulanov Alexander V. , Orlando Mark , West Daniel W. , Moore Daniel R. , Paluska Scott A. , Burd Nicholas A.
Published: November 2023

The consumption of isolated proteins and/or crystalline amino acids enhances the anabolic effect of resistance exercise during recovery. However, the effect of consuming dietary amino acids within their natural nutrient‐dense food matrix to support whole body recovery from resistance exercise has received little attention. Therefore, we determined the effects of whole egg versus egg white ingestion on whole body leucine metabolism in resistance‐trained young males. In crossover trials, six resistance‐trained men (21±1 y; 90±5 kg) received primed constant L‐[1‐13C]leucine infusions with repeated breath and blood samples collected before and 5 h after resistance exercise to determine whole body leucine endogenous rate of appearance (Endo Ra; estimate of protein breakdown), oxidation (OX), non‐oxidative leucine disposal (NOLD; estimate of protein synthesis) and net balance (NB). Immediately after resistance exercise, participants ingested intrinsically L‐[5,5,5‐2H3]leucine labeled whole eggs (18 g protein, 17 g fat; 1.6 g leucine) or egg whites (18 g protein, 0 g fat; 1.6 g leucine) in scrambled form. Endo Ra declined (P<0.001) from fasting values after whole egg (15±1%) and egg white (22±3%) ingestion with no difference between conditions (P=0.14). Similarly, OX decreased over the 5 h period after whole egg and egg white ingestion (P<0.001) with no differences between conditions (P=0.62). NOLD tended to increase (P=0.08) after ingestion of whole egg (10±2%) and egg white (6±3%) with no differences between conditions (P=0.17). As such, NB was negative before exercise but improved similarly (P<0.001) after whole egg (97±6%) and egg white (102±9%) ingestion (P=0.36). In summary, there was a similar increase in whole body NB after the ingestion of whole eggs and egg whites, which was primarily modulated by changes in postprandial whole body protein breakdown rather than protein synthesis during post‐exercise recovery. These data demonstrate that consuming high‐quality protein within a complete food matrix is an effective nutritional strategy to improve whole body net protein balance after resistance exercise in trained young men.

Support or Funding Information

Vision 20/20 Grant, Division of Nutritional Sciences, University of Illinois at Urbana ‐ Champaign
Regulation of Leucine‐induced mTORC1 Activation in Skeletal Muscle of Neonatal Pigs
Authors: Suryawan Agus , Nguyen Hanh V , Parada Rosemarie D , Davis Teresa A
Published: November 2023

Background

Leucine acts as anabolic agent that stimulates protein synthesis in skeletal muscle via activation of mammalian/mechanistic target of rapamycin complex 1 (mTORC1). Studies using cell cultures have proposed several models of the leucine‐induced activation of mTORC1 that involve Sestrin2, leucyl‐tRNA synthetase (LRS), and SH3 domain binding protein 4 (SH3BP4). In the first model, Sestrin2 (a GATOR2‐interacting protein) is a leucine sensor upstream of mTORC1. Lack of leucine causes Sestrin2 to interact with GATOR2, leading to inhibition of mTORC1. Under leucine‐rich condition, the binding of leucine to Sestrin2 disturbs the Sestrin2‐GATOR2 complex resulting in the activation of mTORC1. In a second proposed model, LRS acts as a leucine sensor that positively regulates mTORC1. In this model, there are two proposed pathways involving RagD and Vps34. In the first pathway, leucine binding to LRS stimulates LRS‐RagD interaction, which then promotes activation of mTORC1. In the second pathway, Vps34 mediates amino acid signaling to mTORC1. Leucine‐induced LRS‐Vps34 interaction is necessary for Vps34 activation towards Phospholipase D 1 (PLD1). PLD1 is essential for mTORC1 activation. In the third model, the binding of SH3BP4 to RagB acts as a potent inhibitor of amino acid‐induced activation of mTORC1. Leucine obstructs the interaction of SH3BP4 with RagB, allowing RagB to become fully active and participate in mTORC1 activation.

Objective

We aimed to determine whether these components participate in the leucine‐induced stimulation of mTORC1 in skeletal muscle in vivo.

Methods

Fasted 5‐d‐old piglets were gavage fed every 4 hours either: 1) low protein diet (LP), 2) LP supplemented with Leu (LP+Leu), or 3) high protein diet (HP). Diets were isocaloric and lactose was equal. At 25 h, piglets were injected with L[4‐3H] phenylalanine to measure fractional protein synthesis rates and killed 30 min later. Leucine action toward mTORC1 signaling was determined by measuring mTORC1 activation (mTORC1‐associated mTOR Ser‐2481 autophosphorylation) and the protein‐protein interactions among purported regulatory components of mTORC1 (Sestrin2‐Mios [GATOR2], RagA‐mTOR, RagC‐mTOR, RagB‐SH3BP4, RagD‐LRS, and Vps34‐LRS).

Results

Muscle protein synthesis and mTORC1 activation were higher in the LP+Leu and the HP groups than the LP group. The abundance of the Sestrin2‐GATOR2 complex was lower, and RagA‐mTOR and RagC‐mTOR complexes were greater in the LP+Leu and the HP groups than the LP group. RagB‐SH3BP4, RagD‐LRS, and Vps34‐LRS abundances did not differ among groups.

Conclusion

Sestrin2 and the Rag proteins, but not LRS and SH3BP4, are crucial for the leucine‐induced activation of mTORC1 in skeletal muscle of neonatal pigs.

Support or Funding Information

NIH AR444474, NIH HD085573, NIH HD072891, USDA NIFA 2013‐67015‐20438, and USDA/ARS 6250‐51000‐055.
The Potential of Leucine to Regulate Skeletal Muscle Energy Metabolism Under Obesogenic Conditions
Authors: Baum Jamie I , Inman Thomas G , Shouse Stephanie A
Published: November 2023

Skeletal muscle is a major site of metabolic activity. Healthy skeletal muscle plays a key role in the prevention of metabolic disorders such as obesity and diabetes. Skeletal muscle is responsible for a majority of insulin‐stimulated glucose disposal and plays an important role in the pathogenesis of insulin resistance. Obesity may lead to increased intramyocellular lipid deposition, which is correlated with insulin resistance. In addition, mitochondria play a critical role in energy production and impact whole body metabolic homeostasis. Mitochondrial biogenesis and function are altered with insulin resistance. Mitochondrial dysfunction could be due to deregulated nutrient‐sensing by mammalian target of rapamycin (mTOR), an amino acid sensing pathway. Therefore, the objective of this study was to determine if the branched‐chain amino acid leucine improves mitochondrial function and decreases skeletal muscle fat uptake under obese conditions in skeletal muscle using C2C12 myotubes. C2C12 myotubes were either left untreated (control), treated with 0.75 mM palmitate, 5 mM leucine, or 0.75 mM palmitate + 5 mM leucine for 48 hours. Fifteen minutes before harvesting, cells were treated with or without 100 nM insulin for 15 minutes. Data was analyzed using one‐way ANOVA with the Bonferroni procedure for multiple comparisons. P < 0.05 was considered significant. Treatment with leucine decreased lipid deposition in cells also treated with palmitate, with no additional effect of insulin. There was no effect of palmitate or leucine treatment on PC1a or PPARg expression compared to control cells. However, cells treated with palmitate + Leu + insulin had significantly higher (P < 0.05) PC1a and PPARg expression. There was no treatment effect on Akt phosphorylation compared to controls. These data suggest that leucine has the potential to decrease skeletal muscle fat deposition and improves markers of energy metabolism in obesogenic muscle cells in the presence of insulin.

Support or Funding Information

Arkansas Biosciences Institute. University of Arkansas Honors College Grant.
cAMP‐dependent and independent mitigation of mTORC1‐driven lipogenesis by short‐chain fatty acids, R‐α‐lipoic acid and 4‐phenylbutyric acid
Authors: Moreau Regis , He Bo
Published: November 2023

We used lentiviral delivery of shRNA to knock down TSC2 or Raptor in hepatocellular carcinoma HepG2 cells thereby generating three stable cell lines in which mTORC1 activity is either normal (shScramble cells), low (shRaptor cells), or high (shTSC2 cells). In shTSC2 cells, we previously showed that mTORC1 hyperactivation induced the expression of lipogenic (DGAT1 and DGAT2) and lipoprotein assembly (MTTP and APOB) genes, thereby raising cellular triacylglyceride (TG) levels and exacerbating secretion of apoB‐containing TG‐rich lipoproteins. In this context, two short‐chain fatty acids, R‐α‐lipoic acid (LA) and 4‐phenylbutyric acid (PBA), repressed the secretion of TG‐rich lipoproteins by apparent different mechanisms. The objective of the study was two fold, 1) to determine the individual and combined effects of LA and PBA on markers of lipid metabolism, and 2) to ascertain the mediating role of cAMP in the mechanism of action of LA and PBA. We found that, irrespective of mTORC1 activity level, i) LA or PBA upregulated (P<0.05) the gene expression of CPT1A (LA: +199% in shTSC2 cells; PBA: +184% in shTSC2 cells), ii) LA or PBA downregulated (P<0.05) the gene expression of peroxisomal beta‐oxidation enzyme EHHADH (enoyl‐CoA hydratase/3‐hydroxyacyl‐CoA dehydrogenase (LA: −30% in shTSC2 cells; PBA: −65% in shTSC2 cells), iii) LA, but not PBA, downregulated (P<0.05) the gene expression of DGAT1 (LA: −41% in shTSC2 cells) and DGAT2 (LA: −61% in shTSC2 cells). The combination of LA+PBA further increased CPT1A expression both in shScramble (+180% vs. PBA alone) and shTSC2 (+61% vs. PBA alone) cells, but did not further decrease the expression of EHHADH, DGAT1 or DGAT2 vs. PBA alone. The use of adenylyl cyclase inhibitor, SQ 22536, revealed that cAMP mediates the action of LA, but not that of PBA, by blocking LA‐dependent CPT1A upregulation. We conclude that LA and PBA mitigate mTORC1‐driven lipogenesis by distinct pathways.

Support or Funding Information

Supported by UNL Hatch Act.
Branched chain fatty acids content of natto
Authors: Wang Dong Hao , Yang Yupeng , Lawrence Peter , Brenna J. Thomas
Published: November 2023

Introduction

Branched chain fatty acids (BCFA) are a component of dairy products and beef. The bacterial origin of BCFA suggests they may be rich in fermented foods. We previously found BCFA was at 1% of total fatty acids in sauerkraut and in freshwater fishes. However, dairy products are still the major food source of BCFA, with about 2% BCFA in milkfat, supporting a per capita consumption of 500mg/d BCFA in the United States. In Asian countries where dairy consumption is low, BCFA intake is unlikely to parallel U.S. levels insofar as foods sources are concerned. Natto is a popular Japanese food. It is fermented with bacillus subtilis, which is among species inherently rich in BCFA because of de novo biosynthesized BCFA. Our objective was to determine the BCFA of widely available, traditionally produced natto.

Methods

Two natto products from a Japanese manufacturer were purchased in a local Asian grocery. They were fermented and maintained at refrigerated temperature, and otherwise similar in most ways except being seasoned by different sauces. Total fatty methyl esters were prepared via a modified one‐step hydrolysis procedure and quantified with GC‐FID. BCFA were identified with EIMS/MS.

Results

Natto has 1.6% ± 0.02% BCFA of totally fatty acids, mean of both products. Natto is about 30% calories as fat. Consuming one serving (90 g) of natto would provide 7 g fat and 112 mg of BCFA. The major BCFA in natto are iso‐14:0, iso‐15:0, anteiso‐15:0, iso‐16:0, iso‐17:0 and anteiso‐17:0, substantially recapitulating the BCFA profile of fluid milk except anteiso‐17:0 is lower in natto. Regular consumption of natto in Japan could contribute significant BCFA to overall intake, supplementing Japanese consumption of beef and other known BCFA sources.

Conclusion

Natto may contribute substantially to BCFA consumption where dairy consumption is low.

Support or Funding Information

Internal funds
How Does DHA Reach the Brain to Provide Neuroprotection after Hypoxic‐Ischemic Injury?
Authors: Manual Denny Joseph Kollareth , Zirpoli Hylde , Ten Vadim S. , Deckelbaum Richard Joseph
Published: November 2023

Background

Previous papers have reported that intravenously injected radiolabeled docosahexaenoic acid (DHA) rapidly enters rat brain. Recent studies from our laboratory have shown that acute intraperitoneal (i.p) injection of triglyceride (TG) emulsions rich in n‐3 fatty acids, especially tri‐DHA provides neuroprotection after hypoxic‐ischemic (HI) injury in neonatal mice (PLoS One. 2016;11(8):e0160870).

AIM

To determine how injected tri‐DHA released DHA and perhaps other DHA derivatives are delivered to brain to provide neuroprotection after HI.

Methodology

Laboratory made tri‐DHA emulsions were labeled with non‐metabolizable [3H]‐cholesteryl hexadecyl ether. Neonatal naïve or HI mice (C57BL/6J) were injected with radiolabeled tri‐DHA emulsion intraperitoneally (0.375 mg TG/g bw). At 4 and 24 hrs after injection, blood, peritoneal fluid, lung, liver, heart, brain, kidney, muscle, bone, spleen and adipose tissue were harvested and levels of radiolabeled emulsion in each organ determined. Tissue uptake was expressed as percent of recovered injected dose. HI was produced by right carotid artery ligation followed by hypoxia (8% O2 at 37°C) for 15 mins.

Results

No significant differences were observed between naïve or HI mice in uptake of tri‐DHA emulsion particles at 4 hrs after injection. Among naïve and HI mice, the percent of recovered dose of radioactivity in blood and intraperitoneal fluid at 4 hrs were found to be: blood‐ 7.7 ± 5.5% naïve vs 7.4 ± 0.81% HI; intraperitoneal fluid‐ 12.6 ± 8.8% naïve vs 17.8 ± 7.6% HI. Among all organs assayed, liver contained most of the recovered dose of radiolabeled emulsion (40.5 ± 8.9% naïve vs 42.7 ± 0.1% HI). Lowest emulsion particle uptake was found to be in the brain among naïve and HI mice (<0.1% of the recovered dose among both naïve and HI groups). Compared to 4 hrs, no significant changes were observed in the uptake of emulsion particles at 24 hrs after the injection.

Conclusions

The present data demonstrates that, neonatal mice efficiently clear radiolabeled emulsion from intraperitoneal fluid after i.p. injection. Uptake of emulsion particles was high in liver and very low in brain indicating that whole emulsion particle uptake by brain does not mediate neuroprotection after HI. This suggests the involvement of other pathways in DHA mediated neuroprotection in HI brain. We also hypothesize that local neuroprotection provided by DHA in HI brain is mediated, in part through DHA derivatives produced locally in brain and in other tissues.
Stimulation to the oral cavity increase splanchnic circulation and diet‐induced thermogenesis after drink intake
Authors: Hamada Yuka , Hayashi Naoyuki
Published: November 2023

We recently showed that postprandial splanchnic circulation and diet‐induced thermogenesis (DIT) were greater after slowly eating a test meal than eating the same rapidly (Hamada et al., 2016). It is still unclear whether this is due to masticatory stimulation or the duration of taste stimulation to the oral cavity. The present study examined effects of these stimulations on postprandial splanchnic circulation and DIT. Eleven healthy normal‐weight males (mean±SD: age, 23±1 years; height, 176±4 cm; body mass, 68±5 kg; BMI, 22±1 kg/m2) participated in three trials with a randomized crossover design. A 200‐mL cocoa‐flavored drink (200 kcal) was divided into ten 20‐mL cups. After the baseline measurement for 20 min in the overnight fasting state, the subjects swallowed the ten 20‐mL test drinks over a 5‐min experimental period in three ways on different days. In the control trial (C trial), subjects swallowed one 20‐mL test drink every 30 s. In the long‐duration (taste) stimulation trial (T trial), subjects kept the 20‐mL test drink in their mouth for 30 s without mastication, and then swallowed it (stimulation; control + taste). In the masticatory stimulation trial (M trial), subjects masticated the 20‐mL test drink for 30 s at a frequency of once per second, and then swallowed it (stimulation; control + taste + mastication). Blood flow in celiac artery (CA BF) was calculated from the diameter and the blood velocity. Oxygen uptake was measured until 90 min after the last test drink was swallowed. DIT was calculated from the body mass and increments of oxygen uptake after swallowing the test drink relative to the baseline obtained before the start of each trial. The CA BF accumulated over 90 min after swallowing the test drink was significantly greater in the M trial than in the C trial (mean±SEM: C trial, 3.1±0.5 L; T trial, 3.7±0.6 L; M trial, 4.2±0.8 L). The DIT accumulated over 90 min after swallowing the test drink was significantly greater in the M trial than in the C and T trials (C trial, 43±5 cal/kg; T trial, 75±9 cal/kg; M trial, 107±12 cal/kg). These results suggest that mastication and the duration of taste stimulation to the oral cavity both increase CA BF and DIT.

Support or Funding Information

This work was supported by JSPS KAKENHI Grant Number 15J11944.
Metabolism of Beta‐Alanine in Rat Liver: Degradation to Acetyl‐CoA and Carboxylation to 2‐(aminomethyl)‐malonate
Authors: Wilson Kirkland , Hess Jeremy , Zhang Guofang , Brunengraber Henri , Tochtrop Gregory
Published: November 2023

Beta‐alanine (BA) is a product of uracil and 3‐hydroxypropionate catabolism. BA is a precursor of the dipeptides carnosine and anserine, which play an important role in the pH homeostasis of muscle during exercise. BA is used by athletes as a nutritional supplement to increase performance during strenuous exercise. However, an unwanted side‐effect of the ingestion of BA is unpleasant paresthesia, the mechanism of which is unknown.

As part of a study of 3‐hydroxypropionate metabolism, we perfused rat livers with 2 mM [15N, 13C3]BA (M4 BA). A metabolomic study of the liver showed that acetyl‐CoA was 12% M2 labeled, reflecting oxidation of BA in the citric acid cycle. Urea was 15N‐labeled further confirming the catabolism of BA. We also identified a new compound with a molecular weight equal to BA + 4 + 44. This suggested that BA was carboxylated (MW of CO2 = 44). This was confirmed by perfusing livers with unlabeled BA + either unlabeled NaHCO3 or NaH13CO3; the MW of the new compound was BA + 44 or BA + 45. This confirmed that BA was carboxylated. Based on the labeling and fragmentation patterns, we hypothesized that the compound was 2‐(aminomethyl)‐malonate (AMM). This was confirmed by synthesizing AMM which (after TMS or TBDMS derivatization) had the same mass spectrum and GC‐MS retention time as AMM made from BA in perfused rat livers. Also, direct injection of synthetic AMM into a mass spectrometer source confirmed its MW. AMM was also formed in rat livers perfused with 3‐hydroxypropionate. When 3‐hydroxy‐[13C3]propionate (M3 3HP) was perfused, AMM was M + 3, and M + 2 labeled, confirming the precursor to product relationship.

We showed that the carboxylation of BA is catalyzed by a side‐reaction of pyruvate carboxylase. The enzyme can use either acetyl‐CoA or propionyl‐CoA as an activator. The carboxylation of BA by pyruvate carboxylase is reversible in vitro, like the carboxylation of pyruvate.

In the liver of live rats, the low concentration of AMM was increased by gavage with BA, and further increased by pre‐treatment with aminooxyacetate, a transaminase inhibitor (currently investigated in the treatment of some cancers).

AMM is an isomer of aspartate which is involved in neurotransmission. This raises the possibility that AMM formed after ingestion of BA influences neuronal metabolism, possibly explaining the paresthesia documented with BA ingestion. Also, treatment of some cancers with aminooxyacetate may result in effects similar to BA ingestion.

Support or Funding Information

Supported by the NIH Metabolomic Road Map Initiative
Urinary Enterolignan‐Associated Changes in Plasma Metabolites
Authors: Miles Fayth , Navarro Sandi , Schwarz Yvonne , Randolph Timothy , Neuhouser Marian , Shojaie Ali , Gu Haiwei , Djukovic Danijel , Kratz Mario , Hullar Meredith , Lampe Paul , Raftery Daniel , Lampe Johanna
Published: November 2023

Cancer results from aberrant signaling in biological pathways such as cell proliferation, inflammation and oxidative stress. There is evidence that these pathways may be modified by intake of dietary lignans and their gut bacterial metabolites (enterolignans). We sought to determine if plasma metabolite profiles associated with these pathways differed according to urinary enterolignan excretion in a controlled dietary intervention.

A randomized crossover feeding study (CARB Study) was designed to evaluate effects of two diet patterns: a diet higher in whole grains, legumes, and fresh fruits and vegetables (WG) compared to a diet high in refined grains and added sugars (RG), on biomarkers of cancer‐risk pathways, and was conducted in 80 healthy participants. Fasting blood samples were collected for plasma at baseline and the end of each 28‐d feeding period. Targeted metabolomics was performed on plasma by LC‐MS. 24‐hurinary excretion of enterolignans, enterolactone (ENL) and enterodiol (END), was measured using GC‐MS. Multiple linear regression was performed to measure the association between enterolignan excretion and metabolomic signatures. ENL and END were examined by considering the above or below‐median values specific to the diet pattern, and were additionally analyzed as continuous variables in linear mixed models to examine the effect of enterolignan excretion on metabolite concentrations. Pathway enrichment analyses were conducted using the Global test. Benjamini‐Hochberg false discovery rate (FDR) was used to control for multiple testing.

On average, 65 and 113 μmol/day of total dietary lignans were consumed on the RG, and WG diets, respectively. Of the 175 metabolites assayed, 121 were detected in plasma samples of study participants. Excretion of ENL was associated with differences in 5 metabolites involved in glycine, serine, and threonine metabolism, glycolysis, and energy production; significance was defined as satisfying the FDR of q <0.1. At this FDR level, no metabolites were significantly associated with END excretion. Pathway analysis did not reveal any pathways significantly altered by enterolignan excretion. These results highlight a potential role for ENL produced by the activity of gut bacteria in regulating differentially compounds associated with amino acid and carbohydrate metabolism.

Support or Funding Information

CA192222‐01A1S1
Non‐pungent long chain capsaicin‐analogs arvanil and olvanil display better anti‐invasive activity than capsaicin in human small cell lung cancers
Authors: Colclough Kate Watkins , Seidler Jessica , Akers Austin T , Hurley John D , Brown Kathleen C , Nolan Nicholas A , Dasgupta Piyali
Published: November 2023

The nutritional compound capsaicin inhibits the invasion of many types of human cancers. The clinical development of capsaicin as an anti‐cancer drug is limited due to its unfavorable side effects like burning sensation, stomach cramps, gut pain, and nausea. This study compared the anti‐invasive activity of capsaicin to non‐pungent long chain capsaicin analogs, namely arvanil and olvanil, in human small cell lung cancer cells. Boyden chamber invasion assays revealed that arvanil and olvanil displayed improved anti‐invasive activity relative to capsaicin in human SCLC cells. The results of the Boyden chamber assay were confirmed by the spherical invasion assay, and similar results were obtained. The anti‐invasive activity of arvanil, olvanil and capsaicin were independent of TRPV and CB1 receptors. Furthermore, the anti‐invasive activity of arvanil, olvanil and capsaicin were mediated by the AMPK pathway. Depletion of AMPK levels by siRNA methodology abrogated the anti‐invasive activity of arvanil, olvanil and capsaicin. The non‐pungent capsaicin analogs arvanil and olvanil display improved anti‐invasive activity relative to capsaicin in human SCLC cells. These agents may represent the second generation of capsaicin‐like compounds which are more potent than the parent molecule and have a better side effect profile.

Support or Funding Information

Funding for our study was supported by the an NIH R15‐AREA Grant (2R15CA161491‐02), AICR Investigator Grant, and a NASA Undergraduate Fellowship to JS.
Lysophosphatidic Acid Stimulation of Amphiregulin Shedding Enhanced Paracrine Role of BM CD11+ Cells
Authors: Liu Tianju , De Los Santos Francina Gonzalez , Wu Zhe , Phan Sem H
Published: November 2023

Amphiregulin (AREG), an epidermal growth factor receptor ligand, plays an important role in tissue repair and fibrosis. Bone marrow (BM) CD11c+ cell‐derived AREG promotes pulmonary fibrosis by activating fibroblast proliferation, migration and collagen expression. Pro‐AREG is a membrane bound protein whose mature soluble form represents the extracellular domain that is cleaved off by ADAM17. However, the precise mechanism of regulation or shedding/release of mature AREG is uncertain. Lysophosphatidic acid (LPA) is a bioactive lipid that mediates diverse cellular responses through interactions with specific G‐protein‐coupled receptors. LPA is implicated in tissue repair and wound healing processes, and elevated in the airspaces of patients with idiopathic pulmonary fibrosis, as well as fibrotic lungs of animal model of fibrosis. LPA is known to be able to induce shedding of TGFβ and AREG from cell surface. The objective of this study was to assess the effects of LPA on shedding of AREG ectodomain from CD11c+ cells, and the consequent effect on fibroblast activation. The results showed that LPA caused rapid shedding of AREG from both mouse BM‐derived dendritic cells (BMDC) and human BM CD11c+ mononuclear cells as revealed by the decrease in cell surface AREG expression analyzed by flow cytometry. AREG shedding was confirmed by the elevated level of released AREG protein by LPA‐stimulated BMDC as measured by ELISA. To investigate the paracrine activity of AREG released from BMDC, mouse lung fibroblasts were cultured in the presence of conditioned media from LPA‐stimulated BMDC cultures. The data showed that the fibroblast proliferation rate was significantly increased by these AREG containing conditioned media. These findings together indicated that induced LPA in fibrotic lung tissue could stimulate the shedding of AREG from CD11c+ BM cells, including dendritic cells, and thus enhanced the paracrine role of CD11c+ cells on fibroblast activation in tissue repair and remodeling. They also suggested that targeting the LPA receptor may further mediate its potential therapeutic efficacy for control of tissue fibrosis by reducing AREG shedding.

Support or Funding Information

NIH grants HL052285 and HL112880.