Increase In Lipid Content Research Articles

Analysis of the Effect of Storage Time on Serum Samples for Cholesterol Testing

Cholesterol testing is an important parameter for monitoring lipid metabolism disorders characterized by a decrease or increase in lipid content. The purpose of this study is to determine the effect of storage time on serum samples for cholesterol testing that is delayed by 2 hours and immediate testing. This research was conducted in the laboratory of Puskesmas Tarakan using laboratory observation with the CHOD-PAP method, with the research subjects being students of Politeknik Muhammadiyah Makassar. Based on the cholesterol examination results using 15 samples that were directly tested, there were 3 samples that showed an increase and 12 other samples had normal values. After a 2-hour delay, there was a decrease in all 15 samples. In the paired sample t-test, it can be observed that the average serum sample for direct examination is 170 mg/dl, while the serum sample delayed by 2 hours has an average value of 131 mg/dl, showing a significance of 0.01 < 0.05. Therefore, it can be concluded that there is a significant effect between the direct serum sample and the serum sample delayed by 2 hours. The storage of serum at room temperature must be done immediately to ensure more accurate results and to avoid enzyme changes in the serum.Key words: CHOD-PAP, cholesterol, direct examination.

Lupeol Attenuates Palmitate-Induced Hypertrophy in 3T3-L1 Adipocytes

Obesity is characterized by the enlargement of adipose tissue due to an increased calorie intake exceeding the body’s energy expenditure. Changes in the size of adipose tissue can lead to harmful consequences, with excessive fat accumulation resulting in adipocyte hypertrophy and promoting metabolic dysfunction. These adiposity-associated pathologies can be influenced by dietary components and their potential health benefits. Lupeol, a pharmacologically active pentacyclic triterpenoid found in medicinal plants, vegetables, and fruits, has been shown to exhibit antioxidant and anti-inflammatory properties. This study investigated the role of lupeol on adipocyte hypertrophy by evaluating key adipogenic regulators in vitro. First, 3T3-L1 MBX mouse embryonic cells were differentiated into adipocytes and hypertrophy was induced using 500 µM palmitic acid. The treated adipocytes showed a significantly increased lipid droplet size, confirming adipocyte hypertrophy. Both adipocytes and hypertrophied adipocytes were then treated with or without 60 µM lupeol, following a dose-dependent study. Lipid droplet size was assessed and validated by Oil Red O staining. Western blot analysis was performed to measure the expression of adipogenic and inflammatory markers. Differentiated adipocytes showed increased fatty acid-binding protein 4 (FABP4) expression and Oil Red O staining, indicating an increased lipid content. Western blot analysis revealed that lupeol treatment reduced the expression of FABP4, peroxisome proliferator-activated receptor-γ (PPARγ), and adipokines. In conclusion, the results suggest that lupeol reverts the inflammatory and adipogenic markers that are enhanced in adipocyte hypertrophy. Through its anti-inflammatory effects, lupeol offers protective effects against adipocyte hypertrophy and contributes to reducing hypertrophic adiposity.

Widely targeted metabolomics analysis reveals dynamic changes in metabolites of 'Hass' avocado during postharvest ripening period.

Avocado (Persea americana Mill) is a climacteric fruit harvested at the green stage that ripens postharvest to become edible. The 'Hass' cultivar, known for its rich, buttery flavor and pear-shaped appearance, was studied for metabolite changes during postharvest ripening using UPLC-MS/MS approach. The 16-day ripening period could be divided into three stages: evolution (days 1-9), edible ripe (days 10-15) and overripe (day 16). A total of 1397 metabolites were identified across 13 classes. Among them, amino acids transitioned from conjugated to free forms, while sugars converted to monosaccharides, enhancing savory and sweet tastes. The concentration of certain secondary metabolites like terpenes, coumarins, and alkaloids increased, likely supporting antimicrobial defense, alongside a substantial increase in lipid content. Additionally, 20 compounds were screened as markers for edible ripeness. This study provides valuable insights into avocado postharvest ripening and offers references for optimizing shelf-life.

Amelioration of signaling deficits underlying metabolic shortfall in TREM2R47H human iPSC-derived microglia.

The microglial triggering receptor expressed on myeloid cells 2 (TREM2) is required for diverse microglia responses in neurodegeneration, including immunometabolic plasticity, phagocytosis, and survival. We previously identified that patient iPSC-derived microglia (iPS-Mg) harboring the Alzheimer's disease (AD) TREM2R47H hypomorph display several functional deficits linked to metabolism. To investigate whether these deficits are associated with disruptions in metabolite signaling, we generated common variant, TREM2R47H and TREM2-/- variant human iPS-Mg. We assessed the ability of supplementation with citrate or succinate, key metabolites and cell cycle breaking points upon microglia activation, to overcome these functional deficits with potential impact on neurons. Succinate supplementation was more effective than citrate at overcoming mitochondrial deficits in OXPHOS and did not promote a glycolytic switch. Citrate enhanced the lipid content of TREM2R47H iPS-Mg and was more effective at overcoming Αβ phagocytic deficits, whereas succinate increased lipid content and phagocytic capacity in TREM2-/- iPS-Mg. Microglia cytokine secretion upon pro-inflammatory activation was moderately affected by citrate or succinate showing a condition-dependent increasing trend. Neither metabolite altered basal levels of soluble TREM2 shedding. In addition, neither citrate nor succinate enhanced glycolysis; instead, drove their effects through oxidative phosphorylation. IPS-neurons exposed to conditioned medium from TREM2 variant iPS-Mg showed changes in oxidative phosphorylation, which could be ameliorated when iPS-Mg were first treated with citrate or succinate. Our data point to discrete pathway linkage between microglial metabolism and functional outcomes with implications for AD pathogenesis and treatments.

Exploiting lipid droplet metabolic pathway to foster lipid production: oleosin in focus.

In the past decade, there has been an emerging gap between the demand and supply of vegetable oils globally for both edible and industrial use. Lipids are important biomolecules with enormous applications in the industrial sector and a major source of energy for animals and plants. Hence, to elevate the lipid content through metabolic engineering, new strategies have come up for triacylglycerol (TAG) accumulation and in raising the lipid or oil yield in crop plants. Increased levels of energy density can be achieved by single and multiple gene strategies that re-orient the carbon flux into TAG. Transcription factors and enzymes of the metabolic pathways have been targeted to foster lipid production. Oleosin, a structural protein of the lipid droplet plays a vital role in its stabilization and subsequently in its mobilization for seed germination and seedling growth. Maintenance of increased lipid content with optimal composition is a major target. Knowledge gained from genetic engineering strategiessuggests that oleosin co-expression can result in a significant shift in carbon allocation to LDs. In this review, we present a detailed analysis of the recent advancements in metabolic engineering of plant lipids with emphasis on oleosin with its distinct patterns and functions in plants.

Structural and Physicochemical Properties of Glycerol-Plasticized Edible Films Made from Pea Protein-Based Emulsions Containing Increasing Concentrations of Candelilla Wax or Oleic Acid.

Hydrophobization could improve the moisture resistance of biopolymer-based materials, depending on the methods and materials used, providing benefits for packaging applications. The aim of this study was to compare the effect of increasing concentrations (0-2.0%) of candelilla wax (CW) and oleic acid (OA) on the structural and physicochemical properties, including water affinity, of glycerol-plasticized pea protein isolate (PPI) films. OA acidified the film-forming solution and increased its viscosity more effectively than CW. At the highest concentration, OA prevented cohesive film formation, indicating a weakening of protein self-interaction. OA caused less yellowing, matting, and a smaller reduction in UV/VIS light transmittance compared to CW. Both lipids caused a slight reduction in the films' water content. Phase separation (creaming) of CW enhanced surface hydrophobicity, resulting in a greater reduction in water vapor permeability than OA (~37-63% vs. 2-18%). The addition of lipids did not reduce film solubility or water absorption, and OA even increased these parameters. Increasing lipid content decreased the mechanical strength and stretchability of the films by 28-37% and 18-43%, respectively. The control film exhibited low heat-sealing strength (0.069 N/mm), which improved by 42% and 52% with the addition of CW and OA at optimal levels.

Drying: A Practical Technology for Blueberries (Vaccinium corymbosum L.)-Processes and their Effects on Selected Health-Promoting Properties.

The global dried blueberry market is steadily growing, driven by the creation of innovative blueberry-based products. This trend presents an opportunity to explore a previously untapped segment of the blueberry market in Chile. In this study, a comprehensive assessment of four drying techniques (hot-air drying [HAD], vacuum drying [VD], infrared drying [IRD], and freeze-drying [FD]) was conducted to determine best operating conditions and preserve the health-promoting properties of blueberries. Drying kinetics, proximate composition, color, anthocyanin content, individual phenols, and antioxidant, antiproliferative, and antidiabetic potential of blueberries were evaluated. VD showed the highest drying rates, reaching equilibrium moisture more rapidly (Deff value of 3.44 × 10-10 m2/s). Drying caused an increase in lipid content but a decrease in protein content. The color parameter L* increased in all dried samples, and C* reflected color intensification. FD best retained anthocyanin content, which decreased significantly in the other drying processes. Chlorogenic acid and rutin predominated in HAD, IRD, and FD samples. The antioxidant potential in ORAC assays increased for all drying methods but decreased in DPPH assays. Blueberry extracts from FD and HAD exhibited the greatest antiproliferative effect against A549 and H1299 cell lines, respectively. HAD showed the best inhibitory effect on α-glucosidase, with an IC50 value of 0.276 mg/mL, similar to acarbose (IC50 = 0.253 mg/mL). Given the significant retention of health-promoting properties and bioactive compounds in HAD-dried samples, this method is advisable as a sustainable option for drying blueberries in Chile.

Comparative Analysis of the Biochemical and Molecular Responses of Nannochloropsis gaditana to Nitrogen and Phosphorus Limitation: Phosphorus Limitation Enhances Carotenogenesis.

Nannochloropsis gaditana is well known for its potential for biofuel production due to its high lipid content. Numerous omics and biochemical studies have explored the overall molecular mechanisms underlying the responses of Nannochloropsis sp. to nutrient availability, primarily focusing on lipid metabolism. However, N. gaditana is able to synthesize other valuable products such as carotenoids, including violaxanthin, which has various biological functions and applications. In this study, we comparatively investigated the physiological, biochemical, and molecular responses of N. gaditana to nitrogen and phosphorus limitation, examining biomass production, photosynthetic activity, lipid, chlorophyll, and carotenoids content, and RNA-seq data. Nitrogen limitation decreased photosynthetic activity, chlorophyll content, and biomass production but increased lipid content. Phosphorus limitation substantially increased carotenoids content, with violaxanthin productivity of 10.24 mg/L, 3.38-fold greater than under the control condition, with little effect on biomass production or photosynthetic function. These results were generally consistent with the gene expression pattern observed in transcriptomic analysis. This integrated analysis shows that phosphorus limitation can be an economically competitive solution by enhancing valuable carotenoids while maintaining lipid and biomass production in N. gaditana.

Morphological Alterations and Oxidative Stress Induction in Danio rerio Liver After Short-Term Exposure to the Strobilurin Fungicide Dimoxystrobin

Unlike many other fungicides, strobilurins are applied several times during the growing season for prophylactic purposes, thus heightening the risk of environmental contamination. In the EU, the dimoxystrobin approval period lasted for 17 years. It has been classified as moderately toxic to birds and highly toxic to earthworms, and it is suspected to be carcinogenic to humans. However, it is still commercialized in several countries. The effects of dimoxystrobin are still largely underexplored, with only three studies reporting sublethal alterations in fish. Here, we evaluated for the first time the effects of dimoxystrobin on zebrafish liver after short-term exposure (96 h) to two sublethal and environmentally relevant concentrations (6.56 and 13.13 μg/L), providing evidence of morphological, functional, and ultrastructural modifications. We revealed severe alterations encompassing three reaction patterns: circulatory disturbance, regressive and progressive changes, which also showed a dose-dependent trend. Furthermore, we revealed that dimoxystrobin induced a significant increase in lipid content, a decrease in glycogen granules and affected the defensive response against oxidative stress through a significant downregulation of SOD and CAT. The information presented here demonstrates that the hazardous properties of dimoxystrobin may result from several pathological events involving multiple targets. Our results also emphasize the importance of the combined use of morphological, ultrastructural and functional investigation in ecotoxicological studies.

Influence of Continuous and Short-Term Direct Current Stimulation on the Growth and Nutrient Composition of Nitzschia closterium f. minutissima with Prospects for Large-Scale Production

This study investigates the effects of continuous and short-term direct current (DC) stimulation on the growth and nutrient composition of Nitzschia closterium f. minutissima, a marine diatom with significant potential in aquaculture feed. We explored the optimal conditions of DC stimulation by applying both short-term and continuous treatments at varying voltages (0 V, 1 V, 3 V, 5 V, 10 V for short-term; 0 V, 0.05 V, 0.5 V, 1 V for continuous). The results demonstrated that short-term DC stimulation significantly enhanced cell density and lipid accumulation, with maximum cell density increasing by 13.14% under 1 V stimulation compared to the control. However, continuous stimulation was less effective and showed growth inhibition in several cases. Nutrient analysis revealed that short-term stimulation, particularly at 3 V, led to a 67.66% increase in lipid content, while moderate continuous stimulation (0.5 V) showed a 39.47% increase in biomass dry weight. These findings suggest that short-term DC stimulation is a promising approach to optimize microalgal growth and nutrient accumulation for large-scale aquaculture production.

Ablation of FAM210A in brown adipocytes of mice exacerbates high fat diet induced metabolic dysfunction.

Thermogenesis of brown adipose tissues (BAT) provides metabolic benefits against pathological conditions such as Type 2 diabetes, obesity, cardiovascular diseases, and cancer. The thermogenic function of BAT relies on mitochondria, but whether mitochondrial remodeling is required for the beneficial effects of BAT remains unclear. We have recently identified FAM210A as a BAT-enriched mitochondrial protein essential for cold-induced thermogenesis through the modulation of OPA1-dependent cristae remodeling. Here we report a key role of FAM210A in the systemic response to high-fat diet (HFD). We discovered that HFD suppressed FAM210A expression, associated with excessive OPA1 cleavage in BAT. Ucp1-Cre-driven BAT-specific Fam210a knockout (Fam210aUKO) similarly elevates OPA1 cleavage, accompanied by whitening of BAT. When subjected to HFD, the Fam210aUKO mice gained similar fat mass as sibling control mice, but developed glucose intolerance, insulin resistance, and liver steatosis. The metabolic dysfunction was associated with an overall increased lipid content in both liver and BAT. Additionally, Fam210aUKO leads to inflammation in white adipose tissues. These data demonstrate that FAM210A in BAT is necessary for counteracting HFD-induced metabolic dysfunction but not obesity.

Phytohormone augmented biomass and lipid production in Dunaliella salina under two-stage cultivation strategy: A comprehensive characterization of biomass and lipid using DLS, FTIR, CHNS and NMR for bioenergy prospects

Increasing population, climatic shifts, and decreasing fossil-fuels worldwide led scientists to explore microalgae as a renewable energy resource. The present study unravels the impact of gibberellic acid (GA3) on biomass production, enzymatic activities, macromolecular synthesis, and metabolome analysis of Dunaliella salina using two-stage cultivation strategy. The results indicated a marked increase in biomass (1.44-fold), photosynthetic yield (Fv/Fm 0.68), along with RuBisCO activity (1.66-fold), with an increased in absolute value of zeta potential(ζ) in cultures treated with 10 µM GA3 compared to controls (Stage I). Interestingly, 15 µM GA3-treated cells significantly enhanced lipid content (42.36 %), and carbohydrates (16.56 %) with elevated antioxidative enzymes (SOD, CAT, and APX) activities (Stage II). Enhanced lipid content was further confirmed through FTIR spectra (2928 cm-1, and 1740 cm-1) and 1H NMR signals (1.0– 2, 3.5–4.5 ppm), indicating an increase in both saturated (SFA) and polyunsaturated fatty acids (PUFAs). Elemental analysis demonstrated higher carbon and hydrogen percentages, reflected in higher heating value (HHV) and H/C ratio at 15 µM GA3. The increased lipid content was found to be significantly correlated with ACCase and GPAT activities, suggesting the allocation of carbon flux toward lipid biosynthesis pathways. To substantiate the carbon flux, HR-MS-based metabolomic analysis was performed, indicating a reduction in TCA and Calvin cycle intermediates with a significant rise in carotenoid biosynthesis, as well as metabolic shift towards fatty acid biosynthesis reflecting in the enhanced SFA and PUFAs. Thus, these findings highlighted GA3 significant role in the carbon flux allocation towards the neutral lipid biosynthesis for bioenergy prospects.

Creating of novel Wx allelic variations significantly altering Wx expression and rice eating and cooking quality

Granule-bound starch synthase I (GBSSI) encoding gene Waxy (Wx), which largely regulates the amylose content of rice grains, is a master module determining rice eating and cooking quality (ECQ). Fine-tuning amylose level of grains is an ideal strategy to improve rice quality. Through fine editing of Wxa promoter and 5′UTR by CRISPR/Cas9 system, we created 14 types of novel Wx allelic variations, of which MT7 and MT13 were able to alter Wx expression and amylose content of grains. MT7 showed fragment deletion and base insertions in CAAT-boxes, hardly detectable expression levels of GBSSI mRNA and protein, and generated 5.87% amylose in grains. MT13 had fragment deletions in the A-box and the TATA-box, low expression levels of GBSSI mRNA and protein, and generated 9.61% amylose in grains. Besides of the amylose content, MT7 and MT13 significantly reduced protein content and increased lipid content of grains compared with Wxa. A comparison of MT7, MT13 and other allelic lines demonstrated the importance of base insertion around the second CAAT-box and 31bp-deletion following the second TATA-box in modulating Wx expression. Thus, our study generated two novel Wx allelic variations which significantly alter Wx expression and amylose content of rice grains, providing not only new germplasms for soft rice breeding, but also insights into candidate cis elements of Wx.

Modified natural seawater as growth medium for the halotolerant cyanobacterium Aphanothece halophytica to increase lipid content for biodiesel production

Modified natural seawater as growth medium for the halotolerant cyanobacterium Aphanothece halophytica to increase lipid content for biodiesel production

Combining fermentation broth with spent water recycling for microalgae cultivation: Nutrient reutilization, biodiesel production and techno-economic simulation

This study presents an innovative and promising technology, Water Cycling Combined Fermentation Broth (WC2FB), which offers a sustainable solution to significantly reduce the costs associated with microalgae-based biodiesel production. The innovative approach involves utilizing food waste as a nutrient source, supplemented by recycled water after microalgae harvesting, with the goal of improving the viability of industrial biofuel production. The results of nutrient removal efficiency demonstrated that the addition of 15% fermentation broth to the microalgal suspension efficiently utilized volatile fatty acids, resulting in a significant increase in lipid content. Additionally, biocomponent analysis confirmed that the WC2FB mode did not compromise the desirable properties of biodiesel. Through techno-economic simulations, we predicted a 90% probability of achieving net present value (NPV) at a selling price of $5/kg. Finally, we recommended that directing future efforts be directly towards the development of culture devices, low-cost bioflocculation methods, and biorefinery extraction to further advance biodiesel production.

Abstract 4137414: Uncovering MLKL as a Novel Regulator of Endothelial Cells in the Splenic Niche to Repress Hematopoiesis During Atherosclerosis

Background: Atherosclerosis occurs as lipid and immune cell rich plaques deposit within the arterial wall of the heart. These immune cells are produced from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) and spleen, a process known as hematopoiesis that is dictated by their microenvironment, including endothelial cells (ECs). Objective: While others have shown adverse remodeling of BM ECs during atherogenesis, whether splenic ECs show a similar dysfunction leading to exacerbated hematopoiesis has not been described. Methods: We analyzed RNA sequencing data from chow fed C57Bl/6 mice and high cholesterol diet (HCD) fed Apoe -/- mouse BM and splenic ECs obtained from published studies, as well as our own. In vivo studies were performed in mice on an atherogenic background ( Apoe -/-& Ldlr -/-) fed a HCD for 4 to 16 weeks. Knockdown of the mixed lineage kinase domain-like protein ( Mlkl KD) was achieved by weekly administration of antisense oligonucleotides (ASOs) or scrambled control (50mg/kg sc.). In vitro studies were performed in primary mouse splenic ECs transfected with siRNA. Results: Pathway analysis revealed remarkable tissue-specific responses to an atherogenic milieu, including dysregulation of Lipid Metabolism, Endocytosis and Cell Death pathways in splenic ECs. Interestingly, we previously showed that the mixed lineage kinase domain-like protein (MLKL) regulates the endocytic trafficking of lipids and cell death in macrophages. Indeed using Mlkl KD Apoe -/- mice, transplantation of Mlkl -/- BM into Ldlr -/- mice or EC-specific Mlkl -/- Apoe -/- mice, we show that loss of MLKL drives myelopoiesis and plaque development through regulation of splenic ECs. Both in vivo and in vitro , silencing Mlkl in splenic but not BM ECs potently increased lipid content and disrupted endocytosis by an accumulation of the multivesicular body marker CHMP4B, which mirror the dysregulated pathways we identified above. Furthermore, co-culture with Mlkl KD splenic ECs increased HSPC activation (pStat5) and myeloid colony formation, an effect that was lost when ECs were pre-treated with the upstream endocytosis inhibitor Dynasore. Conclusions: In conclusion, we establish a novel role for MLKL in regulating the balance of HSPCs, specifically through preservation of splenic, but not BM, ECs that repress hematopoiesis, highlighting the importance of splenic lipid metabolism and endocytosis and its impact on atherogenesis.

Impact of an extended light regimen imposed during nursery period on the performance and lipid metabolism of weanling pigs.

This study aimed to assess the impact of a prolonged photoperiod on the growth performance and lipid metabolism of weaned piglets. Twenty-four piglets weaned at 28 days of age were randomly dichotomized into two groups that were alternatively subjected to either long photoperiod (LP) group (16L:8D) or short photoperiod (SP) group (10L:14D) for 42days. Four replicates of three animals per replicates were used per experimental treatment. Our results demonstrated that prolonged photoperiod increased piglet body weight, average daily weight gain (ADG), backfat thickness (BF), backfat index during the nursery period, and increased ADG, average daily feed intake (ADFI), and decreased the F/G of piglets during the experiment days 29 to 42. Meanwhile, we observed LP piglets' plasma melatonin, growth hormone and serotonin levels were decreased at 14 d and 42 d compared to SP piglets. Moreover, up-regulated mRNA or protein expression of PPARγ and CEBPα, and lower mRNA or protein expression of MTR1, ATGL, HSL, PPARα, and CPT1α, were observed in back subcutaneous fat (BSF) of LP group compared with that of SP group. Significant increases were observed in the mRNA or protein contents of lipogenic genes, including C/EBPα, SREBP-1c, ACCα, and FAS, in the liver of LP piglets, whereas CPT1α and ACOX1 mRNA levels and PPARα and MTR1 protein expression were significantly down-regulated in LP group compared to SP group. Extended photoperiod also increased lipid content in longissimus dorsi muscle (LDM) that was associated with higher mRNA or protein levels of SREBP-1c, ACCα, FAS, Pref1, and LPL, decreased mRNA or protein contents of LeptinR, MTR1, HSL, and ACOX1. Together, these findings suggest that there is an advantage, in terms of growth performance and fat deposition, in imposing a prolonged light program (16-h light/day) on nursery piglets to alleviate the negative aspects of weaning stress.

Aligning Advances in Biodiesel Technology with the Needs of the Defense Community

The global transportation sector is transitioning towards renewable energy to combat climate change, with biodiesel playing a critical role. Significant research over the past decades has focused on enhancing biodiesel through novel feedstocks and production methods. The defense community, a major diesel consumer, is particularly interested in biodiesel to support national sustainability goals while also leveraging the benefits of the new technology, including the ability to produce biodiesel locally at the point of need. This paper sets out to review recent advances in biodiesel technology and aligning them with the needs of the defense communities. By doing so, this paper provides insight into the challenges, benefits, and technical feasibility for the two primary consumers of military diesel fuel—naval ships and ground vehicles. For naval applications, algae-based biodiesel shows promise due to its potential for local production near ports. Advances in genetic engineering and cultivation are crucial for increasing lipid content and reducing costs. Innovative methods such as microwave-assisted transesterification and artificial neural networks for optimization could further enhance economic viability. In military ground vehicles, locally produced biodiesel could sustain operations by minimizing supply chain dependencies. Efforts are ongoing to develop mobile production facilities and improve feedstock diversity and methanol-independent transesterification processes. Overall, advancements in biodiesel production from various feedstocks and innovative techniques are poised to significantly benefit the military sector, promoting sustainability and operational efficiency.

Lysate of bovine adipose-derived stem cells accelerates in-vitro development and increases cryotolerance through reduced content of lipid in the in vitro fertilized embryos

Mesenchymal stem cells such as adipose-derived stem cells (ADSCs) are known to secrete factors that stimulate cell division and promote regeneration in neighboring cells. While conditioned medium from stem cells has been used in blastocyst production, no studies have examined the use of cell lysates. In this study we investigated the effects of adding ADSC lysate to in vitro culture (IVC) medium. ADSCs and fibroblasts were isolated from bovine adipose tissue and auricular tissue, respectively, and their lysates were prepared by freeze-thaw disruption. ADSC lysate was added to synthetic oviductal fluid medium. The effects on cleavage, blastocyst development rates, cell numbers, cryotolerance, gene expression (POU5F1, BAX, IGF1R, IGF2R, SOD2), lipid content, and membrane integrity were evaluated according to the experimental design. In Expt. 1, the comparison involved adding ADSC or fibroblast lysate alongside the control group. The total blastocyst rate increased when ADSC lysate was introduced (ADSCs: 40.1 %, fibroblasts: 33.1 %, control: 27.3 %). However, there were no significant differences in the number of trophoblast cells or in the inner cell mass. Experiment 2 confirmed that this increase in blastocyst development was not due to the solvent, PBS(−). In Expt. 3, addition of 10 % fetal calf serum (FCS) or ADSC lysate increased the total blastocyst rate compared to the control (control, 26.2 %; 10 % FCS, 43.4 %; 1 % ADSC lysate, 34.2 %; 10 % ADSC lysate, 48.1 %). After freezing and thawing, the survival and hatching rates of embryos with FCS were significantly lower than those of the control as well as those with added ADSC lysate. In Expt. 4, the addition of ADSC lysate or FCS had no significant effect on gene expression in blastocysts compared to control. However, the addition of FCS significantly increased the gray intensity, indicating higher lipid content compared to the control, with a significant increase in the number of dead cells in the blastocyst. These results indicate that the addition of ADSC lysate to the IVC medium accelerates bovine blastocyst development and that its 10 % addition, corresponding to 1 × 105 cells/mL, is as effective as 10 % FCS without a decrease in cryotolerance due to the increased lipid content.

9210 Nephron specific ATP6AP2 knockout reduces renal fat accumulation in mice consuming high fat diet

Abstract Disclosure: S.A. Culver: None. J. Balugo: None. M. Jansen: None. T. Harris: None. H.M. Siragy: None. Diet induced obesity causes increased renal fat accumulation in both humans and mice which contributes to obesity related kidney injury. We have previously shown that mice with nephron specific ATP6AP2 knockout (KO) consuming high fat diet (HFD) are resistant to weight gain due to increased urinary excretion of glucose, albumin, and fatty acids. Magnetic resonance imaging (MRI) is a useful imaging modality for quantifying organ fat content in live animals though there are few reports of its use quantifying fat in murine kidney. We hypothesized that nephron specific ATP6AP2 knockout inhibits renal fat accumulation with HFD and that we would be able to detect this using MRI. Eight-week-old male wild type (WT) or inducible nephron specific ATP6AP2 KO mice underwent either induction with oral doxycycline or received sucrose water as controls before being placed on either normal diet (ND) (12% fat) or HFD (45% fat) for 3 months. Mice were then imaged using a 9.4 Tesla Bruker BioSpin MRI to determine both liver and kidney cortex fat content. The mean weight of WT HFD mice was 36.03 g compared to 29.03 g in WT ND controls. ND fed and HFD fed KO mice weighed 22.74 g and 23.03 g respectively. MRI showed significant 67% (p<0.05) increase in liver fat content in WT HFD compared to WT ND fed controls, confirming the ability of our MRI technique to detect expected differences in organ fat content. In renal cortex, MRI detected a 54% (p<0.05) increase in lipid content for WT HFD mice compared to WT ND controls. Nephron specific ATP6AP2 KO on HFD demonstrated reduced renal cortical lipid content by 36% (p<0.05) compared to WT HFD controls. These results indicate that ATP6AP2 KO is associated with reduced renal fat accumulation that is measurable with in vivo MRI. MRI therefore represents a valuable tool for studying renal sequelae of obesity in live murine models. Future investigation should examine whether ATP6AP2 KO prevents renal lipotoxicity and kidney injury in the setting of obesity. Presentation: 6/3/2024