bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased circulating trimethylamine N‐oxide, a gut‐flora‐dependent metabolite of choline and betaine in nonalcoholic fatty liver disease is associated with high serum bile acids
Authors: Zhu Hui‐lian , Tan Xu‐ying , Liu Yan , Chen Xiao‐ling
Published: November 2023

Objective

Trimethylamine‐N‐oxide (TMAO) is a gut‐flora‐dependent metabolite of choline and betaine and could dysregulate the bile acids (BAs) metabolism. Many study suggested that elevated circulating TMAO contributes to the risk of cardiovascular diseases, but little is known for non‐alcoholic fatty liver disease (NAFLD). Evidence has linked that elevated circulating bile acids (BAs) level was detrimental to the fatty liver. The pathological mechanisms of TMAO on NAFLD via bile acids are not fully understood. Herein, we examined the association of circulating TMAO, BAs, choline and betaine with the presence and severity of NAFLD in Chinese adults. We also explored correlation of TMAO and BAs and the mRNA expression of bile acid metabolic enzyme.

Methods

We performed a hospital‐based case‐control study. We recruited 60 biopsy‐proven NAFLD cases and 35 controls (18–60 years) and determined serum concentrations of TMAO, BAs, choline and betaine by high‐performance liquid chromatography with online electrospray ionization tandem mass spectrometry (HPLC‐MS/MS). The expression of hepatic bile acids synthesis genes cholesterol 7α‐hydroxylase (CYP7A1) and sterol 27‐hydroxylase (CYP27A1), bile acids transporters genes sodium taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP) were examined by reverse transcriptase‐polymerase chain reaction (RT‐PCR).

Results

In this case control study, serum TMAO increased by 92%, total BAs by 37%, choline by 66.3%, unconjugated BAs by 81%, glycine‐conjugated bile acids (G‐BAs) by 57%, and the ration of betaine /choline decreased by 30.1% in NAFLD patients compared with normal ones and showed significant differences (all P<0.05). Multivariate analyses showed adverse associations of ln‐transformed serum levels of TMAO, total BAs, unconjugated BAs and, G‐BAs and favorable association of betaine/choline ratio with the scores of steatosis and total NAFLD activity (NAS) (all P‐trend <0.05). Circulating TMAO was observed a positively correlated with serum concentration of unconjugated BAs (r=0.306, P=0.022) and G‐BAs (r=0.248, P=0.014). Gene expression of hepatic CYP7A1 were significantly increased (P=0.003) in NAFLD and showed dose‐dependent positive association with serum TMAO (P trend=0.038).

Conclusion

In this case‐control study, our findings suggested adverse associations between the circulating TMAO, choline and BAs level and the presence and severity of NAFLD in Chinese adults. We also found a favorable betaine/choline ration ‐NAFLD relationship. The mechanism of TMAO on NAFLD might be explained by its promoting the expression of bile acid metabolic enzyme of CYP7A1.
Changes in dietary profiles, markers of cardiometabolic health and cognitive function following 12 weeks of peanut consumption
Authors: Coates Alison , Barbour Jayne , Buckley Jonathan , Bryan Janet , Howe Peter
Published: November 2023

Background

Impairments in cardiometabolic health and cognitive function increase with ageing; key dietary macronutrients and micronutrients may counteract these effects. Peanuts contain protein, unsaturated fatty acids, fibre, vitamins and bioactive compounds such as polyphenols that have the potential to improve cardiovascular and cognitive health.

Aim

To investigate the effect of 12 weeks of daily consumption of Hi‐Oleic Australian peanuts compared with a nut free diet on cardiometabolic risk markers and cognitive function.

Methods

Sixty‐one healthy participants (65±7y, BMI 31±4 kg/m2) consumed peanuts (56g/d for 32F; 84g/d for 29M) or no nuts (control) in addition to their habitual diet for 12 weeks each in a randomised crossover design. Diet profiles during each phase were captured using 4‐day weighed food diaries. At baseline and the end of each 12‐week period the following were measured: fasting blood lipids, glucose and insulin, blood pressure, arterial compliance, body composition (DEXA) and inflammation, and cognitive tests were conducted to assess memory, processing speed and executive function.

Results

During the peanut phase compared with the control phase total energy increased by 12% due to higher intakes of protein (12%) and total fat (30%, predominately MUFA). Intakes of fibre (15%), vitamin E (66%), folate (29%) and magnesium (22%) were also increased. Higher MUFA was associated with reductions in fat mass (r=−0.26, p=0.04) and insulin (r=−0.28, p=0.03); higher vitamin E was associated with better large artery elasticity (r=0.27, p=0.04) and higher folate was associated with better memory recall (r=0.35, p=0.007).

Conclusion

Adding Hi‐Oleic peanuts to a nut‐free habitual diet increased consumption of key macronutrients and micronutrients which were associated with improved cardiometabolic risk and memory. Over a longer‐term, peanuts may assist in limiting cardiometabolic risk and cognitive impairments associated with ageing.

Support or Funding Information

Australian Linkage Grant in partnership with the Peanut Company of Australia
Knockdown Glucose‐6‐Phosphate Dehydrogenase promotes EMT through downregulation of E‐cadherin by miR‐200b inhibition in A549 cells
Authors: Wu Yi‐Hsuan , Lee Ying‐Hsuan , Chiu Daniel Tsun‐Yee
Published: November 2023

Glucose‐6‐phosphate dehydrogenase (G6PD) is a house keeping enzyme and is a rate limiting enzyme in the pentose phosphate shunt to regenerate NADPH. Tumorigenesis is often accompanied by increased glycolysis and G6PD activity. However, treatment with TGF‐β, commonly used to induce epithelial‐mesenchymal transition (EMT), inhibits G6PD expression in tumor cells (Accession No. GDS3710). Therefore, the roles of G6PD on tumorigenesis, particularly on EMT need to be clearly defined. In the present study, whether G6PD modulate EMT process in cancer cells through miRNA was evaluated. G6PD‐knockdown cells showed a more mesenchymal‐like morphology. The changes in morphology were accompanied by increase of migration of these cells. Suppression of epithelial markers, E‐cadherin, and increase of N‐cadherin were observed in G6PD‐knockdown cells. The anticancer miRNAs, especially miR‐200b, a regulator of E‐cadherin expression, was downregulated in G6PD‐knockdown cells as documented by miRNA profiling. Ectopic expression of G6PD in G6PD‐knockdown cells upregulates the expression of miR‐200b and E‐cadherin. Taken together, these results suggest that G6PD plays an important role in downregulating EMT via modulation of miR‐200b in lung epithelial cells.

Support or Funding Information

Department of Medical Biotechnology and Laboratory Sciences, College of Medicine, Chang Gung University, Tao‐yuan, Taiwan

Department of Laboratory Medicine, Chang Gung Memorial Hospital, Lin‐Kou, Taiwan
Cystatin Peptide Prevents Alzheimer's Pathology in CRND8 AD Mice
Authors: Shaw Phyllis Ann
Published: November 2023

Alzheimer's disease (AD) is the most common cause of dementia, and upwards of 25 million patients worldwide suffer from AD. The cost of AD care in the United States alone is $214 Billion and expected to reach $1.2 Trillion by 2050 as the population ages. It is thought that AD results from the accumulation of plaques and tangles in the brain leading to eventual neuronal death and cognitive decline. Amyloid‐beta 42 (Aβ42) is the main component of senile plaques. While there are current treatments to address the symptoms associated with AD and cognitive decline, there are no preventative or curative treatments to address the underlying pathophysiology. We identified a peptide capable of binding selectively to Aβ42. In the CRND8 mouse model of AD, intranasal delivery of the peptide reduced the number of Aβ42 plaques by 70–80% in the hippocampus and neocortex compared to scrambled control peptide. Using molecular modeling approaches, a second‐generation peptide was made to improve Aβ42 binding capabilities. The brains of AD mice with no treatment or scrambled peptide treatment had a large accumulation of amyloid beta aggregates and plaque in the neocortex and hippocampus. Importantly, a 4‐week treatment of AD mice with the second‐generation peptide shows a dramatic reduction in the Aβ plaque load in the hippocampus and neocortex. Preliminary behavioral studies suggest that intranasal delivery of the peptide may also improve cognitive function in the Morris Water Maze test. It is proposed that this peptide is a potential disease‐modifying therapy for AD that addresses the underlying pathophysiology.

Support or Funding Information

This work was supported by funds from NIH R21 (AG037448 ) awarded to PAS.
Drug Combinations Modulate Stem Cell Proliferation in the Subventricular Zone of 11‐month‐old Rats: Innate Gender Differences Could Explain Alzheimer's Susceptibility
Authors: Sulehria Tahir , Sharma Neelima , Gagle Samantha , Corbett Adrian M.
Published: November 2023

In this study, we tested the effect of 30 days of various drugs and drug combinations on the stem cell proliferation in the subventricular zone (SVZ) of the lateral brain ventricles of middle aged (10–12 month) normal Sprague Dawley rats, looking for any gender differences. The female rats should be post‐menopausal, and showed very low endogenous stem cell proliferation, significantly different from male rats (P<0.0001). Statins (1 mg/kg) and the combination of Fluoxetine (5 mg/kg), Simvastatin (1 mg/kg) and Ascorbic acid significantly increased stem cell proliferation in the anterior and middle SVZ in female rats, while the three drug combination increased stem cell proliferation in the posterior SVZ. In the male rats, Fluoxetine (5 mg/kg) significantly decreased stem cell proliferation in all regions of the SVZ (anterior, middle, and posterior). Simvastatin (1 mg/kg) significantly decreased stem cell proliferation in the anterior SVZ in male rats, but not in the middle or posterior SVZ. When comparing the stem cell proliferation in different regions across the two genders with a 2‐way ANOVA, we found that fluoxetine and simvastatin significantly decreased male stem cell proliferation compared to females, but the drug combinations of fluoxetine and statin or fluoxetine, statin and ascorbic acid removed these gender differences. The decreased innate stem cell proliferation in females post‐menopause may make them more susceptible to Alzheimer's disease, but we have shown the fluoxetine, simvastatin and ascorbic acid might be used to increase stem cell proliferation, potentially delaying onset of the disease.

Support or Funding Information

WSU Women's in Science Giving Circle
Thymoquinone Enhances Neurogenesis to A Greater Extent in Middle‐Aged Than in Young Aged Rat in Chronic Epilepsy
Authors: Brito Sijam Alassaf , Rao Muddanna Sakattu
Published: November 2023

Neurogenesis in chronic epilepsy is known to be decreased significantly in a young aged, middle aged and aged rats. Thymoquinone (TQ), an active constituent of Nigella sativa seeds possesses anti‐inflammatory, neuroprotective properties and reported to enhance the neurogenesis in different CNS disease models. Aim of the study was to compare the efficacy of TQ in neuro protection immediately after status epileptics (SE), suppression of spontaneous recurrent motor seizures (SRMS) and aberrant mossy sprouting, and enhancement of neurogenesis, in young (4 months) and middle aged (12 months) chronically epileptic rats. SE was induced by intraventricular injection of kainic acid (0.5μg/ventricle) bilaterally in 4 months old and 12 months old male wistar rats(n=12 for each age group). These lesioned rats were treated with TQ (10 mg/kg; ip) for 4 days. A subgroup of lesion only (LO) and lesion+ TQ (L+TQ) rats were sacrificed at 5th day for analysis of hippocampal neurodegeneration by staining the brain sections with flurojade‐B. Remaining rats were video monitored between 2.5–3.0 months post SE for quantifying spontaneous recurrent motor seizure. Lesion only, Lesion+ TQ rats were sacrificed at the end of 3rd month along with normal and sham control rats. (n=6/group). Brain sections were stained with Fluro‐Jade B and labeled for doublecortin. The number newly born neurons and mossy fiber sprouting were quantified in the dentate gyrus at the end of 3rd month. TQ significantly decreased SRMS, reduced the neuron degeneration (25%), in CA1, CA3 and dentate hilus, suppressed the mossy fiber sprouting (30–40%) both in 4 months and 12 months age groups. TQ treatment increased the neurogenesis in both groups, however in middle aged rats it is significantly more than in young aged rats (P<0.001). Our results suggest that TQ is more effective in enhancing the neurogenesis in middle aged chronic epileptic rats than young aged chronic epileptic rats.

Support or Funding Information

(Supported by Kuwait University Grant #YM02/14).
Utility of Virtual 3D Animations and 3D Printed Models of Heart Development in Graduate Embryology Education
Authors: RICHARDSON ALISA , Lee Lisa MJ
Published: November 2023

Visual demonstrations of embryological heart development have typically been presented in 2D images and more recently in scanning electron micrographs. As the heart developmental processes are highly complex both in time and space, the use of 2D visual aids have limitations and are not conducive to effective visualization of the heart looping and septation processes. The objective of this study was to determine the usefulness of virtual 3D animations and tangible, 3D printed heart models in graduate level embryology education. Five 3D virtual heart models were produced using Autodesk MAYA 2016™, each one depicting a different development stage. A virtual 3D animation was created using the virtual models to allow for a 360‐degree turn of each model, in order to provide an accurate depiction of the looping and septation processes. For tangible heart model production, a Markerbot 2 Replicator™ was used to print each virtual model in polylactic acid. The completed virtual animations and 3D printed models were made available to the first year graduate students enrolled in an introductory embryology course. A survey was conducted to gauge students’ perceived value of the virtual and tangible models of embryological heart development. Student exam performance on heart development topics was compared to the previous year's results to quantify educational value of the virtual and 3D printed models. When asked to compare the usefulness of the 3D animations versus the tangible models, students found the animations more useful and conducive to their learning. The quantitative analyses indicate that the 3D virtual animations and the 3D printed models hold promise in facilitating learning of the complex developmental processes in embryology education.

Support or Funding Information

Master of Science in Modern Human Anatomy, University of Colorado School of Medicine
Use of an anatomy eBook to document and discuss anatomical variation
Authors: Thompson Brent J , Barremkala Malli , Raoof Ameed , Attardi Stefanie M , Blenc Ann Marie , Hussein Inaya Hajj , Forbes William , Gould Douglas J , Venuti Judith M
Published: November 2023

In the Gross Anatomy laboratory, students are introduced to the first person that has entrusted them with the care of their body. The laboratory is also an excellent place to learn to appreciate anatomical diversity and the impact that pathological processes have on the body. At Oakland University William Beaumont School of Medicine (OUWB) we are striving to increase the ways students interact with donated cadavers and increase the communication of anatomical and pathological findings with their classmates. To further this effort, we implemented a cadaver assessment form for students to document physical findings, anatomical variations and pathological findings. Utilizing the student observations from these forms, we are building a laboratory photographic eBook record. For example, students have documented anatomical variations such as a third head of the biceps brachii, variations in the arterial branches of the aortic arch, hepatomegaly, splenomegaly and enlarged hearts. They have also found evidence of multiple surgical procedures and cancerous pathologies. At the end of the course, a team of pathologists will participate in a special session to discuss the pathologies found during the semester and their clinical correlates. Students will be surveyed at the end of the semester to evaluate how this project has impacted student communication of anatomical and pathological findings with their classmates.
The effect of the heavy metal nickel on the LINE1 endonuclease
Authors: Smith Michaela , Ahmad Idrees , Meyers Jade , Russell Brittany , Dorrah Kimberly , DeFreece Cecily
Published: November 2023

The long interspersed element‐1 mobile element (LINE1) is a gene that encodes an enzyme protein called LINE1 endonuclease, which is known to nick DNA near adenine and thymine‐rich sites, resulting in double‐strand breaks and the subsequent retrotransposition, or movement, of the element into new genomic loci. Hyperactivity of this element within the human genome has been shown to potentially play a role in cancer‐causing mutations, such as breast and prostate carcinomas. The heavy metals nickel was tested on the LINE1 endonuclease to study the agent's inhibitory effects on the enzyme. Although nickel is a known carcinogen and environmental pollutant, the exact mechanism by which it induces cancer and illness remain unknown. In this experiment, assays were performed on the LINE1 endonuclease, using increasing concentrations of nickel. The data was analyzed using polyacrylamide gel electrophoresis to determine whether an increase in the amount of nickel present, had an effect on LINE1 endonuclease activity. We observed that nickel did not reduce LINE1 endonuclease activity. Resultantly, this could potentially provide a probable explanation for nickel's mechanism of action in certain cancers.

Support or Funding Information

This work was funded by an Institutional Development Award (IDeA) from the NIGMS under grant number P20GM103424 and from the Louisiana Cancer Research Consortium, the NIH‐RCMI grant #8G12MD007595‐05.
Human Milk Oligosaccharides in Preterm and Term Milk
Authors: Morrow Ardythe L , Chen Ceng , Cline Allison , Newburg David S
Published: November 2023

Background

The recommended food of preterm infants is milk from their own mothers, or, if not available, pasteurized donor milk, which typically consists of term milk from late lactation. Human milk oligosaccharides (HMOS) are important for microbial metabolism and infant health. Comparative studies of preterm and term HMOS are needed to guide neonatal nutrition practice.

Objective

To compare the HMOS from Cincinnati, OH mothers of infants who delivered preterm (<29 weeks gestational age [GA]) to those who delivered at term (39–42 weeks GA) in early and late lactation.

Methods

Multiple milk samples were collected from Cincinnati mothers as part of the Global Exploration of Human Milk (GEHM) study, and their HMOS content was analyzed by LC‐MS. Milk collected from 10 to 23 days postpartum (Mo1M) was compared to mature milk (MatM) collected at 3 to 12 months postpartum. All 113 mothers of preterm infants and 118 mothers of term infants provided Mo1M (mean day at collection: 15.2 days – preterm, 16.5 days – term). Of the term mothers, 93 also provided MatM samples that we used to measure donor milk values. Donor milk varies in composition but typically consists of term MM.

Results

Comparing preterm Mo1M to term MatM, composition was similar in total HMOS (3.6 and 3.2 g/L), though the acidic fraction of HMOS was higher in preterm Mo1M than term MatM (1.1 vs 0.6 g/L, p<0.05). But, term Mo1M differed from both preterm Mo1M and term MatM. HMOS concentration of term Mo1M was nearly 2‐fold higher than preterm Mo1M for total HMOS (6.1 vs 3.6 g/L, p<0.001), which held true for both neutral and acidic fractions of HMOS. Significantly (p<0.05) higher values for specific major HMOS were found for term Mo1M compared to preterm Mo1M and term MatM: 2′‐fucosyllactose (2′‐FL, 1.4 vs 0.6 vs 1.0 g/L); 6′‐sialyllactose (6′‐SL, 0.4 vs 0.2 vs 0.1 g/L); and disialyllacto‐N‐tetraose (DSLNT, 0.8 vs 0.2 vs 0.2 g/L). However, some HMOS, e.g., 3′‐sialyllactose (3′‐SL, ~0.2 g/L) maintained similar concentrations regardless of gestational age or timing of lactation.

Conclusions

Preterm milk differs significantly from term milk in HMOS composition in the first month or later lactation, which represents most donor milk. The lower quantity of total HMOS in preterm than term milk should be examined in relation to preterm infant growth and health outcomes.

Support or Funding Information

Supported in part by HD013021 and Mead‐Johnson
Effect of Soy‐Dairy Protein Blend Ingestion on Post‐Exercise Muscle mTORC1 Signaling and Protein Synthesis in Older Adults
Authors: Borack Michael , Reidy Paul , Husaini Syed , Markofski Melissa , Deer Rachel , Richison Abigail , Lambert Bradley , Cope Mark , Mukherjea Ratna , Jennings Kristofer , Volpi Elena , Rasmussen Blake
Published: November 2023

Purpose

Our previous work demonstrated that a soy‐dairy protein blend can prolong aminoacidemia and muscle protein synthesis in young adults following high intensity resistance exercise. The current work investigated the effect of the same soy‐dairy protein blend in older individuals who commonly develop anabolic resistance and sarcopenia. We hypothesized that a soy‐dairy protein blend (whey, soy and casein) would prolong aminoacidemia resulting in enhanced mTORC1 signaling and muscle protein anabolism as compared to a whey protein isolate (WPI) alone.

Methods

The study population for this double‐blind randomized controlled trial included recreationally active men 55–75 years of age. The subjects consumed 30g of WPI or 30g of a soy‐dairy protein blend (25% soy, 25% whey, and 50% casein) one hour following leg extension exercise (8 sets of 10 repetitions at 70% 1RM). Amino acid concentrations as well as basal and post‐exercise muscle protein turnover (breakdown and synthesis) were measured using stable isotopic methods. In addition, skeletal muscle mTORC1 signaling was assessed via western blotting.

Results

There were no differences between the two protein groups for aminoacidemia, mTORC1 signaling or muscle protein turnover following resistance exercise and supplementation. Both groups showed similar blood branched‐chain amino acid profiles following protein ingestion (i.e., leucine, isoleucine, and valine remained elevated (p<0.001) for 240 min) and blood phenylalanine was increased for 185 min in both groups (p<0.001) post‐ingestion. Activation of the mTORC1 pathway (i.e., increased phosphorylation of mTOR, S6K1, and rpS6) was increased to a similar extent in both groups post‐exercise at early (0–2 hrs post‐supplement) and late (2–4 hrs post‐supplement) periods (p<0.05). FBR was significantly reduced post‐exercise for the WPI group (p=0.002) and tended to be reduced in the soy‐dairy protein blend group (P=0.077); however, no difference in post‐exercise FBR was observed between the two groups when controlling for resting FBR and age (p=0.31). Post‐exercise FSR was significantly elevated early for the WPI group (p=0.032), however, post‐exercise FSR did not differ between groups after controlling for resting FSR and age (p=0.38).

Conclusion

Soy‐dairy protein blend induced an increase in aminoacidemia, muscle protein synthesis and mTORC1 signaling to a similar extent as whey protein in older adults when 30 g of protein was ingested after exercise. This data adds new evidence for the use of soy‐dairy protein blends as targeted nutritional interventions that may help overcome anabolic resistance in an effort to counteract sarcopenia.

Support or Funding Information

Grant support provided by DuPont Nutrition & Health
Evaluation of Different Delivery Vehicles on the Digestion and Absorption of Lutein using a Lymph Fistula Rat Model
Authors: Tso Patrick , Vurma Mustafa , Lee Dana , DeMichele Stephen
Published: November 2023

Lutein delivered through breast milk is better absorbed by infants than lutein delivered in infant formula. We wish to better understand the possible absorption differences of lutein in breast milk versus infant formula by determining its bioavailability after gastric administration and to investigate whether the intestinal absorption of lutein can be improved by using new delivery vehicles. In STUDY ONE, we compared, under similar experimental conditions, the intestinal uptake, and the lymphatic and portal transport of lutein in conscious lymph fistula rats. Four groups of rats (n = 8–10/group) were randomized to receive via the gastric tube increasing doses (10 mg/kg, 20 mg/kg, 40 mg/kg, 80 mg/kg) of 20% lutein in safflower oil (SO) suspension to assess whether there was a saturable level of lutein that can be absorbed from the gastrointestinal tract. Aliquots of hourly portal blood and lymph were taken for lutein analysis. The dose response study showed that 20 mg/kg lutein was the saturable level of lymphatic lutein absorption with no lutein detected in portal circulation at any dosage level. In STUDY TWO, we randomized five groups of rats (n = 4–9/group) to receive 20 mg/kg lutein from either lutein in SO or from lutein in four different mono‐diglyceride oils (MDG). Gastric infusion of lutein suspended in MDG (20 mg/kg) significantly improved (71% – 211%; p<0.05) lymphatic lutein output 2–6 hours after lipid feeding versus lutein in SO. We conclude that a mixture of MDG helps solubilize lutein and facilitate gastrointestinal micelle formation thus improving lymphatic lutein absorption compared to triglyceride oils.

Support or Funding Information

DK059630, Abbott Nutrition ZA34B
Potentiation Of TRPM5 Leads To Increased Taste Perception And Insulin Secretion
Authors: Philippaert Koenraad , Vennekens Rudi
Published: November 2023

TRPM5 is a monovalent cation selective ion channel that is activated by raises in the intracellular Ca2+ concentration. This ion channel is functionally expressed in the type II taste receptor cells in the tongue, where it has an essential role in taste signal transduction. TRPM5‐expression is also present in the insulin secreting beta‐cells in the pancreas, where TRPM5 has a regulating role in glucose‐induced insulin secretion. There are currently no good pharmacological tools available to assess the potential of TRPM5 to modulate taste signalling or stimulate insulin secretion.

We show a new class of natural compounds with TRPM5‐potentiating activity. We show potentiation of TRPM5‐mediated current in a heterologous expression system. We observe increased glucose‐induced calcium activity in isolated pancreatic islets of Langerhans when potentiating TRPM5. There is increased insulin secretion in the presence of our potentiator and WT but not TRPM5‐KO mice respond with an increased glucose tolerance during acute and long‐term treatment. Administration of a TRPM5‐potentiator to mice on a high fat diet leads to a healthier glycemic profile and delays or largely prevents the onset of diet‐induced type II diabetes.

In a murine animal model we show stimulation of sweet bitter and umami taste perception. In a two bottle preference test with WT, TRPM5‐KO and sweet receptor‐KO mice, we show that specific potentiation of TRPM5 increases the preferences for sweet solution, without increasing the sugar load.

Our work shows a novel class of compounds to potentiate TRPM5. Stimulating TRPM5 in taste leads to increased perception of nutrients and can lead do a decrease of the calorie intake. We further confirm TRPM5 as a valid pharmacological target in the battle against diabetes to potentiate the physiological profile of glucose induced insulin secretion.
Supplementation of whole flaxseed reduced hepatic steatosis in aged laying hens
Authors: Davis Jeremy E , Cain James , Small Carrie , Hales Dale B
Published: November 2023

Flaxseed has been investigated for its many hepatoprotective properties. Although the essential fatty acid content is often suggested as the underlying mechanism, our recent work as indicated that whole flaxseed may be more protective against the progression of hepatic steatosis. The objective of this study was to determine the effect of feeding different flax‐based diets on the development of fatty liver in aged laying hens—a physiologically relevant model of human liver disease. Laying hens aged three years were fed standard hen diet (CON) or experimental diet supplemented with corn (CRN), fish (FISH), or flaxseed (FXO) oil for 46 weeks. To elucidate the components of flaxseed responsible for hepatoprotective properties, defatted flaxseed (DFM) and whole flaxseed (WFX) meal were also supplemented to experimental diets. At termination, plasma and tissues were collected and analyzed for metabolic and morphologic parameters. Our findings show that WFX supplementation was associated with markedly lower body and liver weight (p< 0.05). This corresponded with a 10–30% reduction in hepatic steatosis (p< 0.05), as well as 40–60% reduction in plasma AST concentration (p< 0.05). Taken together, these data suggest that dietary supplementation with whole flaxseed represents a potential therapy for attenuating the progression of fatty liver.

Support or Funding Information

This work was supported by the following grant NIH/NCCAM RO1AT005295.
Expression and Function of Androgen Receptor in Human Breast Cancer‐Associated Fibroblasts: May Androgens Shape Breast Tumor Microenvironment?
Authors: Lanzino Marilena , Campana Antonella , Giordano Cinzia , Barone Ines , Bonofiglio Daniela , Catalano Stefania , Andò Sebastiano
Published: November 2023

Background

The interaction between breast tumor epithelial and stromal cells is fundamental for tumor growth and progression. Indeed, while providing a scaffold for the breast, the stroma also regulates epithelial cell function through physical and hormonal paracrine exchanges, thus providing a favorable environment for proliferation and metastasis. There is extensive knowledge of androgen receptor (AR) signaling in breast epithelial cancer cells, but less regarding AR‐mediated action in stromal cells of breast tumor microenvironment.

In the present report, we provide evidence of AR expression in breast cancer‐associated fibroblasts (CAFs) isolated from breast cancer patients. We also examined the effect of CAFs exposure to androgens on the migratory behavior of an estrogen‐responsive breast cancer cell line, MCF‐7.

Methods

CAFs were isolated from biopsies of primary breast tumors (n=3); RT‐PCR, immunocytochemistry and immunoblotting analyses were used for evaluating AR expression; CAFs and MCF‐7 breast cancer cells were employed for co‐colture experiments; wound healing and transmigration assays were used for evaluating cell migration.

Results

CAFs do express the AR. RT‐PCR analysis demonstrated the presence of AR mRNA whose expression levels didn't appear to be influenced by administration of 10 nM of the nonmetabolizable synthetic androgen Mibolerone (MIB). Western Blot confirmed the presence, in CAFs lysates, of the AR protein as a ~110kDa protein which was up‐regulated in the presence of MIB, suggesting that AR protein levels might be controlled by post‐traslational mechanisms in CAFs. Immunocytochemical studies further demonstrated that AR is expressed in breast CAFs as a protein with both cytoplasm and nuclear distribution. Immunostaining was more pronounced under MIB treatment, especially in the nucleus.

To explore the relationship between AR expression in the tumor stromal compartment and epithelial cancer cells behavior, co‐colture experiments were carried out by using CAFs and MCF‐7 cells. Interestingly, conditioned medium (CM) from MIB‐treated CAFs was less effective in stimulating MCF‐7 cell motility compared to CM from untreated ones, suggesting the intriguing hypothesis that androgens, via AR, may influence CAFs secretion of paracrine soluble factors involved in tumor cell motility sustainment.

Conclusions

Androgens, via AR, may drive stromal paracrine effects on epithelial breast cancer cells. Disclosing the effectors of androgen response in fibroblast and their impact on tumor microenvironment may provide an alternative perspective on breast cancer progression and therapeutic requirements of patients.

Support or Funding Information

This work was supported by Fondazione Italiana per la Ricerca sul Cancro (FIRC‐AIRC) grants: IG‐11595
Protective Effect of Resveratrol on Mitochondria and Oxidative Stress induced by Cisplatin in HK‐2 Cells
Authors: Valentovic Monica , Murphy Rachel , Lamyaithong Andre Benja , Stafford Reagan , Schnelle Anthony , Ball John G
Published: November 2023

Cisplatin is used in treating many cancers including testicular, ovarian and cervical cancer. Impaired renal function is a major side effect of cisplatin and occurs in 33% of patients treated with cisplatin. Resveratrol (RES) is a polyphenolic compound that possesses anticancer activity. Part of the mechanism for reducing cisplatin toxicity by RES may be mediated by maintaining mitochondrial function and reducing oxidative stress. The purpose of this study was to evaluate the effect of cisplatin on mitochondrial function in a human proximal tubular kidney cell line (HK‐2). Specifically the levels of ATP and ADP were measured following exposure to cisplatin in the presence of RES or DMSO (vehicle). HK‐2 cells were pretreated for 1 h with 0–15 uM RES (vehicle control 10% DMSO). Renal cells were subsequently exposed to cisplatin at a final concentration of 0–30 uM for 24 or 48 h. Viability was assessed using MTT release and cell count with an n=6/treatment group and repeated 3 separate times. Cells pretreated with 10 uM RES were protected from cisplatin cytotoxicity at 24 and 48 h. Cisplatin caused a concentration dependent decline in MTT viability after 24 h exposure to 0, 7.5 and 15 uM cisplatin. RES pretreatment for 1 h with 10 or 15 uM RES increased HK‐2 viability relative to cells exposed only to RES Vehicle (DMSO). Additional studies examined whether RES stimulated cell growth as part of the mechanism for cellular protection. RES did not increase cell number as part of its protective mechanism. ATP levels were diminished by cisplatin causing an increase in the relative balance of ADP/ATP. RES (5 and 7.5 uM) did not alter ATP or ADP/ATP ratios. Additional studies examined Caspase cleavage, protein carbonylation and 4‐hydroxynonenal adduction (4‐HNE) following cisplatin exposure of HK‐2 cells pretreated with DMSO or RES. Western analysis detected an increase in protein carbonylation with 24 h cisplatin exposure which was reversed by RES. In summary, RES protects HK‐2 cells from cisplatin cytotoxicity and preserves mitochondrial ATP levels.

Support or Funding Information

(Supported by NIH Grants INBRE 3P20RR016477‐09S4; 5P20RR016477 and 8P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).
Molecular Mechanisms of All‐Trans‐Retinoic Acid Mediated Cytoprotection Against Renal Cell Injury
Authors: Sapiro Jessica M , Burrell Katherine L , Lau Serrine S
Published: November 2023

One of the major etiologies of acute kidney injury is chemical‐induced nephrotoxicity. Pretreatment of LLC‐PK1 cells with all‐trans‐retinoic acid (ATRA, 25 μM, 24 hr) affords cytoprotection against p‐aminophenol (PAP), and 2‐(glutathion‐S‐yl)hydroquinone‐induced necrosis. In contrast co‐treatment of toxicant with ATRA does not afford cytoprotection, suggesting that ATRA modulates critical signaling pathways prior to toxicant exposure. Interestingly, ATRA did not alter PAP‐induced ROS generation and had a modest effect on Nrf2 induction suggesting other stress response proteins beyond ROS reduction contribute to ATRA protection. Therefore, additional protecting mechanisms were examined. During ATRA‐mediated pretreatment, the ER stress proteins Grp78 and p‐eIF2α increased 2‐fold in a time‐dependent manner (ATRA 24 hr and 4 hr, respectively). Furthermore, ATRA increased expression of the cellular stress kinases p‐ERK and p‐AKT with maximum levels at 30 min. p‐ERK decreased to basal levels after 4 hr, while p‐AKT remained elevated for 24 hr, coinciding with the time required for maximal ATRA cytoprotection. Moreover, induction of these stress kinases was observed at concentrations of ATRA (10 and 25 μM) required for cytoprotection. Interestingly, the p‐ERK inhibitor PD98059 reduced ATRA protection against PAP toxicity indicating a possible function of p‐ERK and its downstream target genes in the ATRA mechanism for protection. We further determined that ATRA led to a prompt increase in cyclin D1 levels (1–4 hr, 3‐fold). Collectively, UPR proteins (Grp78, p‐eIF2α), stress kinases (p‐AKT, p‐ERK) and mitogenic effects (cyclin D1) function interdependently to afford ATRA protection against renal cell damage

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ES006694, T32ES007091, ES016578, ASPET‐SURF
Cytochrome P450 enzymes involved in the primary metabolism of the novel constitutive androstane receptor inhibitor, CINPA1
Authors: Cherian Milu T , Yang Lei , Chai Sergio C , Lin Wenwei , Chen Taosheng
Published: November 2023

This study aims to identify the metabolic enzymes involved in the breakdown of CINPA1, a recently discovered small molecule inhibitor of the xenobiotic receptor CAR (constitutive androstane receptor).

CAR and PXR are xenobiotic sensors that respond to drugs and endobiotics by modulating the expression of metabolic genes that enhance detoxification and elimination. Elevated levels of drug metabolizing Phase I, Phase II enzymes and efflux transporters resulting from CAR activation promote the elimination of chemotherapeutic agents leading to reduced therapeutic effectiveness. Multidrug resistance in tumors after chemotherapy could be associated with errant CAR activity, as shown in the case of neuroblastoma. CAR inhibitors used in combination with existing chemotherapeutics could be utilized to attenuate multidrug resistance and resensitize chemo‐resistant cancer cells. But such studies have been confounded so far as previously reported CAR inhibitors are also activators of pregnane X receptor (PXR). This renders them mechanistically counterproductive since CAR and PXR have overlapping functions. Through a directed screening approach we identified new CAR inhibitors from a subset of small molecules shortlisted as PXR inhibitors in a high‐throughput screen of 320,000 chemicals.

The lead small molecule discovered was CINPA1, capable of reducing CAR‐mediated transcription with an IC50 of ~70 nM but without activating PXR. The mechanisms by which CINPA1 inhibits CAR function were also extensively examined and elucidated. CINPA1 association with the CAR‐LBD reduced recruitment of coactivators. Reduced CAR occupancy at the promoter regions of its target genes was observed in the presence of CINPA1. The pharmacokinetic properties of CINPA1 were determined and CINPA1 confirmed to be broken down into two main metabolites. In vitro enzyme assays analyzed by LC‐MS facilitated the identification of the cytochrome P450 enzymes responsible for metabolism of CINPA1 in human liver microsomes. CINPA1 is converted to the first metabolite by CYP3A4 and then further metabolized by CYP2D6. The metabolic profiling information for CINPA1 will help guide the in vivo PK/PD studies in mice. Ongoing structure activity relationship studies have also revealed some key features of this inhibitor and metabolites. Metabolite 1 is a mild CAR inhibitor while metabolite 2 has no effect on CAR function. Computational modeling using the CAR‐LBD structure shows that while CINPA1 and metabolite 1 can bind in the CAR ligand binding pocket, metabolite 2 might be unable to make the necessary molecular interactions required for binding.

We describe the pharmacokinetic properties of CINPA1, a potent inhibitor of CAR that could be a novel molecular tool for understanding CAR function and could be utilized for resensitizing chemoresistant cancer cells as in the case of neuroblastoma.

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NIH, ALSAC
Nitric Oxide Stimulates CYP51A1 Down Regulation
Authors: Byrd Aria , Lee Choon‐myung , Park Ji Won , Morgan Edward Thomas
Published: November 2023

Cytochrome P450 proteins (P450) are a family of enzymes found in virtually all living organisms. These proteins are responsible for the metabolism of xenobiotics as well as clearance and biosynthesis of endogenous compounds in the body. Cholesterol is an endogenous sterol that is vital for maintaining cell membrane fluidity and serving as a precursor for the production of important biological compounds such as vitamin D, steroid hormones and bile acids. The production of cholesterol is reliant on CYP51A1, which is the only P450 that converts lanosterol to 4, 4‐dimethylcholesta‐8(9), 14, 24‐trien‐3β‐ol via 14α‐demethylation. CYP51A1 is also believed to be the oldest recognizable P450, as it is the only P450 with a conserved function across animal, fungal and plant kingdoms. Previous studies have shown that nitric oxide (NO) formed in hepatocytes in response to inflammatory stimuli can cause down regulation of a number of drug metabolizing P450s. The objective of this study is to determine if CYP51A1 is down regulated by NO. Huh7 cell lines were transduced with lentiviral vectors containing pLX304‐CYP51A1V5 to generate v5‐tagged CYP51A1 expressing cell lines. HeLa cell lines were transduced with lentivirus containing doxycycline inducible pLIX304‐hNOS2, in which human nitric oxide synthase2 (hNOS2) is regulated through a tetracycline promoter region, and additionally transduced with lentiviral vectors containing pLX304‐CYP51A1V5. Huh7 cell lines were treated with NO donors (the NONOates DPTA and NOC‐18, and S‐nitrosoglutathione, GSNO) and then CYP51A1 protein expression was determined by Western Blot Analysis. Results showed that NOC‐18 down regulated 51A1 in both a time and dose dependent manner. Treatment with DPTA (6 h) and NOC‐18 (18 h) were more effective than GSNO in down regulation of 51A1. The HeLa cell lines treated with doxycycline produced amounts of NO comparable to those observed in human hepatocytes, which was blocked by the competitive hNOS2 inhibitor L‐NG‐nitroarginine methyl ester (L‐NAME). Doxycycline treatment for 12 or 18 hours down regulated 51A1 protein expression to 25–30% of control values. L‐NAME treatment blocked the down‐regulation of CYP51A1 in doxycycline‐treated cells. We conclude that NO stimulates the down regulation of CYP51A1 in both HeLa and Huh7 cell lines. Future studies will investigate whether this down regulation is achieved through proteosomal degradation subsequent to ubiquitin tagging, as has been seen in the down regulation of drug metabolizing P450s like CYP2B1.

Support or Funding Information

This research is supported by the National Institutes of Health grant R01GM069971
Synergistic Interaction of Histone Deacetylase Inhibitors with Interferon‐β in Inducing Apoptosis of Human Neuroblastoma Cells
Authors: Dedoni Simona , Olianas Maria C. , Onali Pierluigi
Published: November 2023

The type I interferon (IFN) family of cytokines, comprising IFN‐α, IFN‐β, IFN‐ω and IFN‐κ, displays antiproliferative activity and has been used with some success in the treatment of different human neoplasias. We have previously observed that IFN‐β causes cell death of human SH‐SY5Y neuroblastoma (NB) cells by inducing apoptosis through the intrinsic mitochondrial pathway (Dedoni et al. J. Neurochem. 115,1421‐1433, 2010). The histone deacetylase (HDAC) inhibitor valproic acid has been reported to enhance the antiproliferative effects of type I IFNs, but little is known on whether the two agents synergize in inducing NB cell apoptosis. In the present study we show that prolonged exposure (24 h) of SH‐SY5Y cells to therapeutic concentrations of valproic acid (0.6 and 1 mM) had no effect on cell viability but, when combined with IFN‐β (30 ng/ml), significantly enhanced the apoptotic cell death induced by the cytokine. The synergistic interaction was associated with enhanced expression of apoptotic markers, including caspase activation, DNA fragmentation, and cleavage of the DNA‐repairing enzyme poly‐(ADP ribose) polymerase (PARP) and the pro‐survival transcription factor β‐catenin. Valproic acid enhanced the IFN‐β‐stimulated Tyr‐phosphorylation of STAT1, which is required by the cytokine to trigger apoptosis. Sodium butyrate (1 mM) and trichostatin (100 and 300 nM), two additional HDAC inhibitors, mimicked the valproic acid potentiation of IFN‐β‐induced apoptosis, whereas valpromide (1 mM), which did not enhance histone acetylation, was without effect. Differently from SH‐SY5Y cells, MYCN‐amplified human LAN‐1 NB cells displayed no sensitivity to the pro‐apoptotic activity of IFN‐β, but underwent apoptosis when treated with either valproic acid or sodium butyrate. Co‐treatment of LAN‐1 cells with IFN‐β and HDAC inhibitors, but not valpromide, resulted in a significant increase in the expression of the apoptotic markers. These data demonstrate a synergistic interaction of IFN‐β and HDAC inhibitors in promoting the apoptotic death of NB cells and support the use of this combination for the treatment of human NB.

Support or Funding Information

The study was supported by Regione Autonoma della Sardegna, Italy, L.R. n. 7/2007‐CRP10810/2012.
Diminazene Aceturate Attenuates Obesity and Promotes a Balance of the Renin‐Angiotensin System in Fat and Muscle Tissue in Young and Aged Rats on High Fat Diet
Authors: Sakarya Yasemin , Bruce Erin , Morgan Drake , Tümer Nihal , Carter Christy S. , Scarpace Philip J.
Published: November 2023

Obesity is a complex disorder, and increases the risk for a number of chronic diseases, including diabetes and cardiovascular diseases. Renin‐angiotensin system (RAS) is considered the major regulator of cardiovascular homeostasis. Recent studies suggest stimulation of the vasoprotective ACE2/Ang (1–7)/Mas axis of the RAS leads to significant reductions in body weight in obese animal models. We have shown that Diminazene Aceturate, a novel ACE2 activator, significantly reduced body weight of both young and aged rats fed 60% high fat diet. Notably we observed a significant loss of fat mass with a preservation of lean mass. We hypothesize that maintaining balance between the ACE/AngII/AT1R axis and vasoprotective components in muscle and white adipose tissues will prevent much of the associated pathophysiology of the obesity.

Male Fisher 344 × Brown Norway rats, ages 4 (n=12) and 23 months (n=17) were fed 60% high fat diet for one week, whereupon, animals were further divided, and given either 15mg/kg/day DIZE s.c. (Young DIZE, n=6; Old DIZE, n=9) or vehicle (Young Control, n=6; Old Control, n=8). Body weight and food intake were measured throughout the experiment. Time Domain‐Nuclear Magnetic Resonance (TD‐NMR) was used to assess body composition 1 week and 3 weeks after the start of DIZE treatment. With the aim of elucidating the effect of DIZE on the relationship between RAS components, AT1R, AT2R, MasR, ACE, ACE2 mRNA were measured in retroperitoneal white adipose tissue (RTWAT), and tibialis anterior (TA) muscle, using qPCR.

Relative quantitation of RTWAT mRNA expression showed DIZE treatment significantly increased AT2R (5 fold) and MasR (1 fold) in old animals when compared to non‐treated controls. AT1R expression was significantly increased in the muscle of old rats treated with DIZE; however, the AT1R/AT2R ratio was not different from control. Furthermore, the ACE/ACE2 ratio was decreased in the muscle tissue in both young and old rats. Our data suggests DIZE may be a useful tool in maintaining the balance between the two opposing arms of the RAS, and will be an effective treatment to ameliorate obesity and related disorders.

Support or Funding Information

Hypertension Center T32 Post Doctoral training grant, North Florida/South Georgia Veterans Health System (Research/GRECC), and National Institute's of Health Grant NIH DK 091710