bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

BackgroundLeucine 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.ObjectiveWe aimed to determine whether these components participate in the leucine‐induced stimulation of mTORC1 in skeletal muscle in vivo.MethodsFasted 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).ResultsMuscle 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.ConclusionSestrin2 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 InformationNIH 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 InformationArkansas 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 InformationSupported 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

IntroductionBranched 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.MethodsTwo 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.ResultsNatto 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.ConclusionNatto may contribute substantially to BCFA consumption where dairy consumption is low.Support or Funding InformationInternal 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

BackgroundPrevious 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).AIMTo determine how injected tri‐DHA released DHA and perhaps other DHA derivatives are delivered to brain to provide neuroprotection after HI.MethodologyLaboratory 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.ResultsNo 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.ConclusionsThe 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 InformationThis 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 InformationSupported 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 InformationCA192222‐01A1S1
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 InformationFunding for our study was supported by the an NIH R15‐AREA Grant (2R15CA161491‐02), AICR Investigator Grant, and a NASA Undergraduate Fellowship to JS.