Lipid Droplet Area Research Articles

Feline cumulus cells and oocytes show massive accumulation of lipid droplets and upregulation of PLIN2 expression after in vitro maturation

This study investigates lipid content and expression of genes involved in lipid metabolism in feline cumulus cells and oocytes before and after in vitro maturation (IVM). Domestic cats represent valuable models in reproductive research, yet the efficiency of in vitro embryo production remains suboptimal, with contributing factors still under investigation. We characterized lipid droplets (LDs) in oocytes collected from adult queens, both before and after IVM, using confocal microscopy with Bodipy 493/503 staining. We also quantified the expression of four genes involved in regulation of lipid metabolism. Our findings revealed a substantial accumulation of LDs and a significant upregulation of PLIN2 expression in both cumulus cells and oocytes following IVM. The number and total area of LDs and fluorescence intensity increased markedly in oocytes at the MII stage, while the average LD diameter decreased. Similarly, cumulus cells showed an increase in number and total area of LDs post-IVM, suggesting their involvement in oocyte maturation by modulating lipid homeostasis. This study presents the first detailed characterization of lipid droplet dynamics in feline oocytes and cumulus cells, providing insights into metabolic processes during IVM, which are critical for optimizing assisted reproductive technologies in felids.

Label-free long-term measurements of adipocyte differentiation from patient-driven fibroblasts and quantitative analyses of in situ lipid droplet generation

The visualization and tracking of adipocytes and their lipid droplets (LDs) during differentiation are pivotal in developmental biology and regenerative medicine studies. Traditional staining or labeling methods, however, pose significant challenges due to their labor-intensive sample preparation, potential disruption of intrinsic cellular physiology, and limited observation timeframe. This study introduces a novel method for long-term visualization and quantification of biophysical parameters of LDs in unlabeled adipocytes, utilizing the refractive index (RI) distributions of LDs and cells. We employ low-coherence holotomography (HT) to systematically investigate and quantitatively analyze the 42-day redifferentiation process of fat cells into adipocytes. This technique yields three-dimensional, high-resolution refractive tomograms of adipocytes, enabling precise segmentation of LDs based on their elevated RI values. Subsequent automated analysis quantifies the mean concentration, volume, projected area, and dry mass of individual LDs, revealing a gradual increase corresponding with adipocyte maturation. Our findings demonstrate that HT is a potent tool for non-invasively monitoring live adipocyte differentiation and analyzing LD accumulation. This study, therefore, offers valuable insights into adipogenesis and lipid research, establishing HT and image-based analysis as a promising approach in these fields.

Gestational Diabetes-like Fuels Impair Mitochondrial Function and Long-Chain Fatty Acid Uptake in Human Trophoblasts.

In the parent, gestational diabetes mellitus (GDM) causes both hyperglycemia and hyperlipidemia. Despite excess lipid availability, infants exposed to GDM are at risk for essential long-chain polyunsaturated fatty acid (LCPUFA) deficiency. Isotope studies have confirmed less LCPUFA transfer from the parent to the fetus, but how diabetic fuels impact placental fatty acid (FA) uptake and lipid droplet partitioning is not well-understood. We evaluated the effects of high glucose conditions, high lipid conditions, and their combination on trophoblast growth, viability, mitochondrial bioenergetics, BODIPY-labeled fatty acid (FA) uptake, and lipid droplet dynamics. The addition of four carbons or one double bond to FA acyl chains dramatically affected the uptake in both BeWo and primary isolated cytotrophoblasts (CTBs). The uptake was further impacted by media exposure. The combination-exposed trophoblasts had more mitochondrial protein (p = 0.01), but impaired maximal and spare respiratory capacities (p < 0.001 and p < 0.0001), as well as lower viability (p = 0.004), due to apoptosis. The combination-exposed trophoblasts had unimpaired uptake of BODIPY C12 but had significantly less whole-cell and lipid droplet uptake of BODIPY C16, with an altered lipid droplet count, area, and subcellular localization, whereas these differences were not seen with individual high glucose or lipid exposure. These findings bring us closer to understanding how GDM perturbs active FA transport to increase the risk of adverse outcomes from placental and neonatal lipid accumulation alongside LCPUFA deficiency.

Female-age-dependent changes in the lipid fingerprint of the mammalian oocytes.

Can oocyte functionality be assessed by observing changes in their intracytoplasmic lipid droplets (LDs) profiles? Lipid profile changes can reliably be detected in human oocytes; lipid changes are linked with maternal age and impaired developmental competence in a mouse model. In all cellular components, lipid damage is the earliest manifestation of oxidative stress (OS), which leads to a cascade of negative consequences for organelles and DNA. Lipid damage is marked by the accumulation of LDs. We hypothesized that impaired oocyte functionality resulting from aging and associated OS could be assessed by changes in LDs profile, hereafter called lipid fingerprint (LF). To investigate if it is possible to detect differences in oocyte LF, we subjected human GV-stage oocytes to spectroscopic examinations. For this, a total of 48 oocytes derived from 26 young healthy women (under 33 years of age) with no history of infertility, enrolled in an oocyte donation program, were analyzed. Furthermore, 30 GV human oocytes from 12 women were analyzed by transmission electron microscopy (TEM). To evaluate the effect of oocytes' lipid profile changes on embryo development, a total of 52 C57BL/6 wild-type mice and 125 Gnpat+/- mice were also used. Human oocytes were assessed by label-free cell imaging via coherent anti-Stokes Raman spectroscopy (CARS). Further confirmation of LF changes was conducted using spontaneous Raman followed by Fourier transform infrared (FTIR) spectroscopies and TEM. Additionally, to evaluate whether LF changes are associated with developmental competence, mouse oocytes and blastocysts were evaluated using TEM and the lipid dyes BODIPY and Nile Red. Mouse embryonic exosomes were evaluated using flow cytometry, FTIR and FT-Raman spectroscopies. Here we demonstrated progressive changes in the LF of oocytes associated with the woman's age consisting of increased LDs size, area, and number. LF variations in oocytes were detectable also within individual donors. This finding makes LF assessment a promising tool to grade oocytes of the same patient, based on their quality. We next demonstrated age-associated changes in oocytes reflected by lipid peroxidation and composition changes; the accumulation of carotenoids; and alterations of structural properties of lipid bilayers. Finally, using a mouse model, we showed that LF changes in oocytes are negatively associated with the secretion of embryonic exosomes prior to implantation. Deficient exosome secretion disrupts communication between the embryo and the uterus and thus may explain recurrent implantation failures in advanced-age patients. Due to differences in lipid content between different species' oocytes, the developmental impact of lipid oxidation and consequent LF changes may differ across mammalian oocytes. Our findings open the possibility to develop an innovative tool for oocyte assessment and highlight likely functional connections between oocyte LDs and embryonic exosome secretion. By recognizing the role of oocyte LF in shaping the embryo's ability to implant, our original work points to future directions of research relevant to developmental biology and reproductive medicine. This research was funded by National Science Centre of Poland, Grants: 2021/41/B/NZ3/03507 and 2019/35/B/NZ4/03547 (to G.E.P.); 2022/44/C/NZ4/00076 (to M.F.H.) and 2019/35/N/NZ3/03213 (to Ł.G.). M.F.H. is a National Agency for Academic Exchange (NAWA) fellow (GA ULM/2019/1/00097/U/00001). K.F. is a Diamond Grant fellow (Ministry of Education and Science GA 0175/DIA/2019/28). The open-access publication of this article was funded by the Priority Research Area BioS under the program "Excellence Initiative - Research University" at the Jagiellonian University in Krakow. The authors declare no competing interest. N/A.

Quantitative assessment of morphological changes in lipid droplets and lipid-mito interactions with aging in brown adipose.

The physical characteristics of brown adipose tissue (BAT) are defined by the presence of multilocular lipid droplets (LDs) within the brown adipocytes and a high abundance of iron-containing mitochondria, which give it its characteristic color. Normal mitochondrial function is, in part, regulated by organelle-to-organelle contacts. For example, the contact sites that mediate mitochondria-LD interactions are thought to have various physiological roles, such as the synthesis and metabolism of lipids. Aging is associated with mitochondrial dysfunction, and previous studies show that there are changes in mitochondrial structure and the proteins that modulate organelle contact sites. However, how mitochondria-LD interactions change with aging has yet to be fully clarified. Therefore, we sought to define age-related changes in LD morphology and mitochondria-lipid interactions in BAT. We examined the three-dimensional morphology of mitochondria and LDs in young (3-month) and aged (2-year) murine BAT using serial block face-scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. Our analyses showed reductions in LD volume, area, and perimeter in aged samples in comparison to young samples. Additionally, we observed changes in LD appearance and type in aged samples compared to young samples. Notably, we found differences in mitochondrial interactions with LDs, which could implicate that these contacts may be important for energetics in aging. Upon further investigation, we also found changes in mitochondrial and cristae structure for the mitochondria interacting with LDs. Overall, these data define the nature of LD morphology and organelle-organelle contacts during aging and provide insight into LD contact site changes that interconnect biogerontology with mitochondrial function, metabolism, and bioactivity in aged BAT.

Utilizing superheated steam to prepare traditional Chinese twice-cooked pork bellies, exploring its effects on the texture and flavor of fat layers

Fat greatly impacts the overall texture and flavor of pork belly. Twice-cooked pork bellies (TPB), typically boiled and sliced before “back to pot” being stir-fried, is a classic Sichuan cuisine among stir-fried dishes. In this study, the effects of substituting conventional pan-frying (PCV) with superheated steam (SHS) technology on the sensory, texture, microstructure and flavor of the fat layers were investigated. SHS was used as an alternative to boiling (120 °C for 15, 20, 25, and 30 min), and “back to pot” stir-frying was also by SHS. TPB precooked for 25 min (P25) with SHS performed better quality characteristics than PCV, with less collagen fiber disruption and lipid droplet area, resulting in a lower hardness and higher shear force. Besides, the low-oxygen environment of SHS retarded the lipid peroxidation, showing a significantly lower MDA content than PCV. Differently, PCV exhibited more grassy and fatty flavors, while P25 exhibited a unique aroma of fruity and creamy due to its higher UFA/SFA ratios in the pre-cooking stage. Overall, the sensory scores of P25 were comparable to those of PCV (with no significant difference), revealing that SHS is expected to be applied to the industrial production of stir-fried dishes.

O-102 From oocytes to embryos

Abstract Title: Oxidative stress and DNA damage in oocytes: clinical relevance and diagnosis Study question: Is it possible to detect signs of oxidative stress (OS) in a living oocyte? Summary answer: OS-driven changes in lipid profile can be detected in human oocytes. What is known already: Oxidative stress and DNA damage are associated with increasing age of women. From all cellular molecules, lipids are most prone to OS-driven damage. DNA is more stable, so its damage may occur only later, probably as a result of cytoplasmic changes caused by damage to lipids and proteins. Initially, peroxidation of cellular lipids causes damage to lipid bilayers i.e. biological membranes and membranous organelles. Accumulation of lipid peroxidation products - toxic aldehydes - causes DNA damage and mutations. Screening of DNA damage in the oocyte is not feasible in human ART. However, oocyte lipid damage can be detected using live-cell label-free spectroscopies. So far, changes in the lipid profile of human oocytes have not been investigated. Here we propose the screening of lipid profile as a reliable method of evaluation of OS-driven damage of the oocyte. Study design, size, duration: This study aims to thoroughly discuss effects of OS in female gamete. Special consideration is given to current limits in DNA damage detection and oocyte quality screening. Then, strategies to evaluate OS-driven damage of cellular components are proposed. Finally, obstacles and future perspectives in clinical diagnosis and treatment of OS-damaged oocytes are discussed. Participants/materials, setting, methods: To investigate if it is possible to detect differences in oocyte LF, we examined a total of 48 GV oocytes derived from 26 women under 33 years of age and with no history of infertility, enrolled in an oocyte donation program. Furthermore, to evaluate the effect of lipid profile changes in oocytes on embryo development, 167 C57BL/6 mice were used. Modifications of cytoplasmic lipid droplets (LDs) profile in oocytes were evaluated by Coherent anti-stokes Raman Spectroscopy (CARS) and further validated by Fourier Transform Infrared Spectroscopy (FTIR) and Transmission Electron Microscopy (TEM). The influence of oocyte lipid changes on embryo development was verified using mouse model. Main results and the role of chance: Here we show woman-age-dependent progressive changes in LF of oocytes consisting of increased LDs size, area and number, assessed by label-free imaging (CARS). LF variations in oocytes were detectable also within individual donors. We next confirmed woman-age-dependent changes in oocytes by demonstration of lipid peroxidation and composition changes (FTIR) and alterations of structural properties of lipid bilayers (TEM). Finally, using a mouse model we showed that LF changes in oocytes negatively affect preimplantation embryo development. Limitations, reasons for caution: The idea of this study was to propose an original approach to assess the quality of oocytes based on changes in the lipid profile. The information presented should not be viewed as directly applicable, because human oocytes at the GV stage were assessed, and not at the MII stage. Wider implications of the findings: Our findings highlight functional connections between lipids and other molecules building the oocyte, including DNA. Furthermore, this original study open the possibility to develop an innovative tool for oocyte assessment. Study funding/competing interest(s): This research was funded by National Science Centre of Poland Grants 2021/41/B/NZ3/03507 and 2019/35/B/NZ4/03547.

The autophagic regulation of rosiglitazone-promoted adipocyte browning.

Objective: Browning of white adipocytes is considered an efficient approach to combat obesity. Rosiglitazone induces the thermogenetic program of white adipocytes, but the underlying mechanisms remain elusive. Methods: Expression levels of browning and autophagy flux markers were detected by real-time PCR and immunoblotting. H&E and Oil Red O staining were performed to evaluate the lipid droplets area. Nuclear protein extraction and immunoprecipitation were used to detect the proteins interaction. Results: In this study, we reported that rosiglitazone promoted adipocyte browning and inhibited autophagy. Rapamycin, an autophagy inducer, reversed adipocyte browning induced by rosiglitazone. Autophagy inhibition by rosiglitazone does not prevent mitochondrial clearance, which was considered to promote adipose whitening. Instead, autophagy inhibition increased p62 nuclear translocation and stabilized the PPARγ-RXRα heterodimer, which is an essential transcription factor for adipocyte browning. We found that rosiglitazone activated NRF2 in mature adipocytes. Inhibition of NRF2 by ML385 reversed autophagy inhibition and the pro-browning effect of rosiglitazone. Conclusion: Our study linked autophagy inhibition with rosiglitazone-promoted browning of adipocytes and provided a mechanistic insight into the pharmacological effects of rosiglitazone.

Milk fat globule membrane proteins are crucial in regulating lipid digestion during simulated in vitro infant gastrointestinal digestion

Products of lipolysis released during digestion positively affect the metabolism of newborns. In contrast to the 3-layer biological membranes covering human milk (HM) fat, the lipid droplets in infant milk formula (IMF) are covered by a single membrane composed of casein and whey proteins. To reduce the differences in lipid structure between IMF and HM, studies have used milk fat globule membrane (MFGM) components such as milk polar lipids (MPL) to prepare emulsions mimicking HM fat globules However, few studies have elucidated the effect of membrane proteins (MP) on lipid digestion in infants. In this study, 3 kinds of emulsions were prepared: One with MPL as the interfaced of lipid droplets (RE-1), one with membrane protein concentrate (MPC) (RE-2) as the interface of lipid droplets, and one with both MPL and MPC (1:2) as the co-interface of lipid droplets (RE-3). The interfacial coverage of the emulsions was confirmed by measuring the contents of MPL and MPC at the lipid droplet interface, and by confocal laser scanning microscopy analyzed. By controlling the homogenization intensity, the specific surface area of lipid droplets was controlled at the same level among the 3 emulsions. The stability constants of the emulsions varied, and RE-1 was the most stable. During simulated in vitro infant gastrointestinal digestion, the amount of free fatty acids (FFA) released from the lipid droplets was significantly higher from those with MPC at the interface (RE-2, RE-3) than from that with MPL at the interface (RE-1). The amount of FFA released at the end of intestinal digestion of RE-1, RE-2, and RE-3 was 255.00 ± 3.54 µmol,328.75 ± 5.30 µmol, 298.50 ± 9.19 µmol, respectively. Compared with the lipid droplets in RE-2, those with MPL at the interface (RE-1, RE-3) released more unsaturated fatty acids (USFAs) during digestion. The emulsifying activity index was highest in RE-3 (MPL and MPC co-interface). The presence of MPL at the emulsion interface increased the release of USFAs, while the presence of MPC increased the release of FFA. These results show that both MPL and MP are indispensable in the construction of MFGM. Understanding their effects on digestion can provide new strategies for the development of infant foods.

Quantitative Assessment of Morphological Changes in Lipid Droplets and Lipid-Mito Interactions with Aging in Brown Adipose.

The physical characteristics of brown adipose tissue (BAT) are defined by the presence of multilocular lipid droplets (LD) within the brown adipocytes and a high abundance of iron-containing mitochondria, which give it its characteristic color. Normal mitochondrial function is, in part, regulated by organelle-to-organelle contacts. Particularly, the contact sites that mediate mitochondria-LD interactions are thought to have various physiological roles, such as the synthesis and metabolism of lipids. Aging is associated with mitochondrial dysfunction, and previous studies show that there are changes in mitochondrial structure and proteins that modulate organelle contact sites. However, how mitochondria-LD interactions change with aging has yet to be fully clarified. Therefore, we sought to define age-related changes in LD morphology and mitochondria-lipid interactions in BAT. We examined the three-dimensional morphology of mitochondria and LDs in young (3-month) and aged (2-year) murine BAT using serial block face-scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. Analysis showed reductions in LD volume, area, and perimeter in aged samples compared to young samples. Additionally, we observed changes in LD appearance and type in aged samples compared to young samples. Notably, we found differences in mitochondrial interactions with LDs, which could implicate that these contacts may be important for energetics in aging. Upon further investigation, we also found changes in mitochondrial and cristae structure for mitochondria interacting with LD lipids. Overall, these data define the nature of LD morphology and organelle-organelle contacts during aging and provide insight into LD contact site changes that interconnect biogerontology and mitochondrial functionality, metabolism, and bioactivity in aged BAT.

Myofiber “Lipotoxicity” Does Not Disrupt Cellular Biophysical Properties

Obesity is associated with pathological changes in skeletal muscle that contribute to functional loss and disability. While it is unclear which specific factors cause this impairment in muscle performance, intramyocellular lipid (IMCL) within the muscle fibers, termed “lipotoxicity”, is implicated. Some studies have suggested the mechanism for this association is mitochondrial dysfunction, whereas others propose the increase in IMCL leads to structural changes within the muscle fibers. The objective of this study was to determine whether IMCL, independent of obesity, impacted active force generation and passive viscoelasticity of the myofiber sarcomere lattice. This study utilized soleus muscles from wildtype (WT) and lipodystrophic (LD) mice and calf muscle biopsies from individuals with (DPN) and without (CTL) type 2 diabetes over a range of obesity status to isolate the effects of IMCL from the effects of obesity in general. IMCL was quantified for each fiber type by co-registration of Oil Red O (ORO) stained cryosections or fluorescent BODIPY stained section and myosin heavy chain isoform stained sections. BODIPY stained sections were used with confocal microscopy to quantify individual lipid droplet metrics (average area, total number). Individual muscle fibers were isolated from soleus muscles and calf muscle biopsies and chemically permeabilized to isolate the myofibrillar lattice and were tested in a specialized system (1400A; Aurora Scientific). Active and passive forces were recorded, fit with a 3-element model, and correlated with metrics of IMCL accumulation by fiber type. LD muscle had 2-3 fold higher IMCL accumulation (p&lt;0.05) in type 2a and 2x fibers than WT quantified by ORO and BODIPY. This was due to an increase in both lipid droplet area and number. DPN muscle had ~2 fold higher IMCL accumulation (p&lt;0.05) in type 2 fibers compared with CTL. Both type 1 and type 2 fibers had significantly increased area of lipid droplets, but only in type 2 fibers was there an increase in lipid droplet number and consequently in area fraction. Despite the large accumulation of IMCL, LD permeabilized muscle fibers had no deficit in active force generation and no change in any of the 5 measures of passive viscoelasticity compared with WT. Conversely, in humans, DPN type 2 fibers had significantly lower (-18%) active force generation and both type 1 and type 2 DPN fibers had elevated resting viscosity (+5%) compared with CTL. Across human samples, active force generation was significantly correlated with average lipid droplet area (r2=0.53, p=0.04), but no other correlations were significant. This study suggests that an increase in IMCL accumulation in and of itself does not physically disrupt sarcomere contraction. Dramatic accumulation of IMCL in LD mouse fibers did not impact active or passive forces, likely due to an absence of systemic complications (obesity, advanced diabetes). Human fibers of comparable IMCL accumulation have a deficit in active force generation correlated with IMCL parameters, but we conclude this relationship is more likely correlative than causative as type 1 fibers with similar lipid droplet areas have no association between lipid droplet area and active force generation. These data suggest that while accumulation of IMCL is associated with contractile disfunction across a number of conditions, the mechanism is likely either indirect or biochemical rather than biophysical. Shriners Hospitals for Children, P30 AR074992, AR075773, AG15768, AG46927, AR072999, AR073752. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Impact of Exposure to a Mixture of Organophosphate Esters on Adrenal Cell Phenotype, Lipidome, and Function.

Organophosphate esters (OPEs) are used primarily as flame retardants and plasticizers. Previously, we reported that adrenal cells are important targets of individual OPEs. However, real-life exposures are to complex mixtures of these chemicals. To address this, we exposed H295R human adrenal cells to varying dilutions (1/1000K to 1/3K) of a Canadian household dust-based OPE mixture for 48 hours and evaluated effects on phenotypic, lipidomic, and functional parameters. Using a high-content screening approach, we assessed phenotypic markers at mixture concentrations at which there was greater than 70% cell survival; the most striking effect of the OPE mixture was a 2.5-fold increase in the total area of lipid droplets. We then determined the response of specific lipid species to OPE exposures with novel, nontargeted lipidomic analysis of isolated lipid droplets. These data revealed that house dust OPEs induced concentration-dependent alterations in the composition of lipid droplets, particularly affecting the triglyceride, diglyceride, phosphatidylcholine, and cholesterol ester subclasses. The steroid-producing function of adrenal cells in the presence or absence of a steroidogenic stimulus, forskolin, was determined. While the production of 17β-estradiol remained unaffected, a slight decrease in testosterone production was observed after stimulation. Conversely, a 2-fold increase in both basal and stimulated cortisol and aldosterone production was observed. Thus, exposure to a house dust-based mixture of OPEs exerts endocrine-disrupting effects on adrenal cells, highlighting the importance of assessing the effects of environmentally relevant mixtures.

SARS-CoV-2 Infection Alters the Phenotype and Gene Expression of Adipocytes.

Epidemiological evidence emphasizes that excess fat mass is associated with an increased risk of severe COVID-19 disease. Nevertheless, the intricate interplay between SARS-CoV-2 and adipocytes remains poorly understood. It is crucial to decipher the progression of COVID-19 both in the acute phase and on long-term outcomes. In this study, an in vitro model using the human SGBS cell line (Simpson-Golabi-Behmel syndrome) was developed to investigate the infectivity of SARS-CoV-2 in adipocytes, and the effects of virus exposure on adipocyte function. Our results show that SGBS adipocytes expressing ACE2 are susceptible to SARS-CoV-2 infection, as evidenced by the release of the viral genome into the medium, detection of the nucleocapsid in cell lysates, and positive immunostaining for the spike protein. Infected adipocytes show remarkable changes compared to uninfected controls: increased surface area of lipid droplets, upregulated expression of genes of inflammation (Haptoglobin, MCP-1, IL-6, PAI-1), increased oxidative stress (MnSOD), and a concomitant reduction of transcripts related to adipocyte function (leptin, fatty acid synthase, perilipin). Moreover, exogenous expression of spike protein in SGBS adipocytes also led to an increase in lipid droplet size. In conclusion using the human SGBS cell line, we detected SARS-CoV-2 infectivity in adipocytes, revealing substantial morphological and functional changes in infected cells.

Free Radical-Mediated Photocyclization of Triphenylphosphindole Oxides for Photoactivated and Self-Reported Lipid Peroxidation.

Photocyclization is demonstrated as a powerful tool for building complicated polycyclic molecules. And efficient photocyclization is competent as an artful strategy to develop photo-responsive smart materials. Herein, an efficient free radical-mediated photocyclization for triphenylphosphindole oxide (TPPIO) derivatives to generate tribenzophosphindole oxide (TBPIO) derivatives at ambient condition is reported. The reaction mechanism and substituent effect on photocyclization efficiency are thoroughly investigated. Additionally, photophysical and photochemical properties of TPPIO and TBPIO derivatives are measured for comparison and deeply deciphered by theoretical calculation. TPPIO derivatives own typical aggregation-induced emission feature but barely generate reactive oxygen species (ROS), while TBPIO derivatives experience aggregation-caused quenching but show efficient Type I ROS generation capacity. Further, in vitro experiments demonstrate that this photo-conversion can efficiently occur in situ in living cells to activate photodynamic therapy (PDT) effect to trigger lipid peroxidation with selective fluorescence "light up" in lipid droplet area under continuous irradiation. This work extends the optoelectronically and biologically interesting phosphindole oxide-containing π-conjugated systems through an efficient synthetic strategy, provides in-depth mechanistic descriptions in the aspects of reaction and property, and further presents their great potentials for photoactivated and self-reported PDT.

Lycopene Alleviates the Adverse Effects of Feeding High-Lipid Diets to Hybrid Grouper (♀Epinephelus fuscoguttatus ×♂E. lanceolatus).

It has been found that high-lipid diets (HLDs) disrupt lipid metabolism in fish, leading to an excessive accumulation of lipids in various tissues of the fish body. The objective of this study was to investigate if the inclusion of lycopene (LCP) in an HLD may mitigate the adverse consequences of excessive dietary lipid intake in hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ E. lanceolatus). The experimental design incorporated a control group (L0), which was administered a diet consisting of 42% protein and 16% lipid. The diets for groups L1, L2, and L3 were developed by augmenting the control diet with 100, 200, and 400 mg/kg LCP, respectively. The duration of the trial spanned a period of 42 days. The results of the study showed that the weight gain rate (WGR) and protein efficiency ratio (PER) of the three LCP treatment groups (L1, L2, and L3) tended to increase and then decrease, with a significant increase in WGR and PER in L2 (P < 0.05). Visceral somatic index and hepatic somatic index tended to decrease and then increase in all treatment groups, with a significant decrease in L2 (P < 0.05). In serum dietary LCP significantly reduced triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL) content and significantly increased high-density lipoprotein (HDL) content (P < 0.05). In the liver, dietary LCP reduced TC, TG, and very LDL levels and improved lipoprotein lipase, hepatic lipase, fatty acid (FA) synthetase, and acetyl-CoA carboxylase activities. The number and area of hepatic lipid droplets decreased significantly with increasing LCP content. In the liver, the addition of appropriate levels of LCP significantly upregulated lipoprotein lipase (lpl) and peroxisome proliferator-activated receptor α (pparα). In summary, dietary LCP improves growth and reduces lipid deposition in the liver of hybrid grouper by increasing lipolytic metabolism and decreasing FA synthesis. Under the experimental conditions, the fitted curve analysis showed that the recommended LCP additions to the high lipid diet for juvenile hybrid grouper were 200-300 mg/kg.

PACAP regulates VPAC1 expression, inflammatory processes and lipid homeostasis in M1- and M2-macrophages.

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts as an anti-atherogenic neuropeptide and plays an important role in cytoprotective, as well as inflammatory processes, and cardiovascular regulation. Therefore, the aim of this study is to investigate the regulatory effects of PACAP and its receptor VPAC1 in relation to inflammatory processes and lipid homeostasis in different macrophage (MΦ) subtypes. To investigate the role of PACAP deficiency in the pathogenesis of atherosclerosis under standard chow (SC) or cholesterol-enriched diet (CED) in vivo, PACAP-/- mice were crossbred with ApoE-/- to generate PACAP-/-/ApoE-/- mice. Lumen stenosis in the aortic arch and different MΦ-subtypes were analyzed in atherosclerotic plaques by quantitative immunohistochemistry. Undifferentiated bone marrow-derived cells (BMDC) from 30-weeks-old ApoE-/- and PACAP-/-/ApoE-/- mice were isolated, differentiated into BMDM1- and BMDM2-MΦ, and incubated with oxidized low-density lipoprotein (oxLDL). In addition, PMA-differentiated human THP-1 MΦ were further differentiated into M1-/M2-MΦ and subsequently treated with PACAP38, the VPAC1 agonist [(Ala11,22,28)VIP], the antagonist (PG 97-269), and/or oxLDL. Uptake/accumulation of oxLDL was analyzed by oxLDL-DyLight™488 and Bodipy™ 493/503. The mRNA expression was analyzed by qRT-PCR, protein levels by Western blot, and cytokine release by ELISA. In vivo, after 30 weeks of SC, PACAP-/-/ApoE-/- mice showed increased lumen stenosis compared with ApoE-/- mice. In atherosclerotic plaques of PACAP-/-/ApoE-/- mice under CED, immunoreactive areas of VPAC1, CD86, and CD163 were increased compared with ApoE-/- mice. In vitro, VPAC1 protein levels were increased in PACAP-/-/ApoE-/- BMDM compared with ApoE-/- BMDM, resulting in increased TNF-α mRNA expression in BMDM1-MΦ and decreased TNF-α release in BMDM2-MΦ. Concerning lipid homeostasis, PACAP deficiency decreased the area of lipid droplets in BMDM1-/M2-MΦ with concomitant increasing adipose differentiation-related protein level. In THP-1 M1-/M2-MΦ, the VPAC1 antagonist increased the uptake of oxLDL, whereas the VPAC1 agonist decreased the oxLDL-induced intracellular triglyceride content. Our data suggest that PACAP via VPAC1 signaling plays an important regulatory role in inflammatory processes in atherosclerotic plaques and in lipid homeostasis in different MΦ-subtypes, thereby affecting foam cell formation. Therefore, VPAC1 agonists or PACAP may represent a new class of anti-atherogenic therapeutics.

The roles of DGAT1 and DGAT2 in human myotubes are dependent on donor patho-physiological background.

The roles of DGAT1 and DGAT2 in lipid metabolism and insulin responsiveness of human skeletal muscle were studied using cryosections and myotubes prepared from muscle biopsies from control, athlete, and impaired glucose regulation (IGR) cohorts of men. The previously observed increases in intramuscular triacylglycerol (IMTG) in athletes and IGR were shown to be related to an increase in lipid droplet (LD) area in type I fibers in athletes but, conversely, in type II fibers in IGR subjects. Specific inhibition of both diacylglycerol acyltransferase (DGAT) 1 and 2 decreased fatty acid (FA) uptake by myotubes, whereas only DGAT2 inhibition also decreased fatty acid oxidation. Fatty acid uptake in myotubes was negatively correlated with the lactate thresholds of the respective donors. DGAT2 inhibition lowered acetate uptake and oxidation in myotubes from all cohorts whereas DGAT1 inhibition had no effect. A positive correlation between acetate oxidation in myotubes and resting metabolic rate (RMR) from fatty acid oxidation in vivo was observed. Myotubes from athletes and IGR had higher rates of de novo lipogenesis from acetate that were normalized by DGAT2 inhibition. Moreover, DGAT2 inhibition in myotubes also resulted in increased insulin-induced Akt phosphorylation. The differential effects of DGAT1 and DGAT2 inhibition suggest that the specialized role of DGAT2 in esterifying nascent diacylglycerols and de novo synthesized FA is associated with synthesis of a pool of triacylglycerol, which upon hydrolysis results in effectors that promote mitochondrial fatty acid oxidation but decrease insulin signaling in skeletal muscle cells.

Transcriptional Knock-down of mstn Encoding Myostatin Improves Muscle Quality of Nile Tilapia (Oreochromis niloticus)

Myostatin (encoded by mstn) negatively regulates skeletal muscle mass and affects lipid metabolism. To explore the regulatory effects of mstn on muscle development and lipid metabolism in Nile tilapia (Oreochromis niloticus), we used antisense RNA to transcriptionally knock-down mstn. At 180 days, the body weight and body length were significantly higher in the mstn-knock-down group than in the control group (p < 0.05). Additionally, fish with mstn-knock-down exhibited myofiber hyperplasia but not hypertrophy. Oil red O staining revealed a remarkable increase in the area of lipid droplets in muscle in the mstn-knockdown group (p < 0.05). Nutrient composition analyses of muscle tissue showed that the crude fat content was significantly increased in the mstn-knock-down group (p < 0.05). The contents of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids were all significantly increased in the mstn-knock-down group (p < 0.05). Comparative transcriptome analyses revealed 2420 significant differentially expressed genes between the mstn-knock-down group and the control group. KEGG analysis indicates that disruptions to fatty acid degradation, glycerolipid metabolism, and the PPAR signaling pathway affect muscle development and lipid metabolism in mstn-knock-down Nile tilapia: acaa2, eci1, and lepr were remarkably up-regulated, and acadvl, lpl, foxo3, myod1, myog, and myf5 were significantly down-regulated (p < 0.05). These results show that knock-down of mstn results in abnormal lipid metabolism, acceleration of skeletal muscle development, and increased adipogenesis and weight gain in Nile tilapia.

Comparative study of carbohydrate levels on growth, oxidative stress and glucolipid metabolism of hybrid fish between Megalobrama amblycephala (♀) × Culter alburnus (♂) and Culter alburnus

The present study used hybrid fish (Megalobrama amblycephala ♀ × Culter alburnus ♂, abbreviated as BT) and topmouth culter (Culter alburnus, abbreviated as TC) as research subjects to assess and compare the effects of carbohydrate on the growth, oxidative stress and glucolipid metabolism of the two fish. The TC (initial weight, 13.10 ± 0.10 g) and BT (initial weight, 12.85 ± 0.13 g) were fed isonitrogenous (43% crude protein) and isolipidic (6.5% crude lipid) diets with carbohydrate levels of 10% (LC diet) and 30% (HC diet) for 11 weeks, respectively. The results showed that the HC diet resulted in a significant decrease in specific growth rate (SGR) of both fish. However, the SGR in BT was significantly higher than in TC, regardless of diet. Furthermore, HC diet significantly increased serum alanine transaminase (GPT) and hepatic malondialdehyde (MDA) content, decreased hepatic catalase (CAT) activity and resulted in increased hepatocyte volume, nucleus deficiency and nucleus deviation from the center of cytoplasm in both fish. However, BT had significantly higher glutathione peroxidase (GPx) activity and lower serum GPT and aspartate transaminase (GOT) content compared to TC. The HC diet induced significant increases in hepatosomatic index (HSI), liver lipid droplet area, liver glycogen, serum glucose (GLU) and triglyceride (TG) content in both fish. However, HSI and lipid droplet area were significantly higher in BT than in TC, while serum GLU and TG content were significantly lower in BT than in TC. Both fish can adapt to the HC diet by up-regulating the expression of glycolysis-related gene (glucokinase (gk)) and lipogenesis-related genes (acetyl-CoA carboxylase a (acca), fatty acid synthase (fas)). However, the expression levels of glucose transporter 2 (glut2), glycolysis-related gene (phosphofructokinase liver b (pfklb)) and lipogenesis-related genes (sterol regulatory element-binding protein 1 (srebp1), ATP citrate lyase a (aclya)) were increased only in BT fed HC diet. In summary, the BT liver has stronger glucose transport, glycolysis, and lipid tolerance compared to TC, thus exhibiting better glucose homeostasis and growth performance. It indicates that hybridization may help to produce new strain of fish with high carbohydrate utilization capacity.

Investigating ionizing radiation-induced changes in breast cancer cells using stimulated Raman scattering microscopy.

Altered lipid metabolism of cancer cells has been implicated in increased radiation resistance. A better understanding of this phenomenon may lead to improved radiation treatment planning. Stimulated Raman scattering (SRS) microscopy enables label-free and quantitative imaging of cellular lipids but has never been applied in this domain. We sought to investigate the radiobiological response in human breast cancer MCF7 cells using SRS microscopy, focusing on how radiation affects lipid droplet (LD) distribution and cellular morphology. MCF7 breast cancer cells were exposed to either 0 or 30Gy (X-ray) ionizing radiation and imaged using a spectrally focused SRS microscope every 24hrs over a 72-hr time period. Images were analyzed to quantify changes in LD area per cell, lipid and protein content per cell, and cellular morphology. Cell viability and confluency were measured using a live cell imaging system while radiation-induced lipid peroxidation was assessed using BODIPY C11 staining and flow cytometry. The LD area per cell and total lipid and protein intensities per cell were found to increase significantly for irradiated cells compared to control cells from 48 to 72hrs post irradiation. Increased cell size, vacuole formation, and multinucleation were observed as well. No significant cell death was observed due to irradiation, but lipid peroxidation was found to be greater in the irradiated cells than control cells at 72hrs. This pilot study demonstrates the potential of SRS imaging for investigating ionizing radiation-induced changes in cancer cells without the use of fluorescent labels.